Devices and Methods for Sample Analysis

ABSTRACT

Methods, devices, and systems for analyte analysis using a nanopore are disclosed. The methods, devices, and systems utilize a first and a second binding member that each specifically bind to an analyte in a biological sample. The method further includes detecting and/or counting a cleavable tag attached to the second binding member and correlating the presence and/or the number of tags to presence and/or concentration of the analyte. Certain aspects of the methods do not involve a tag, rather the second binding member may be directly detected/quantitated. The detecting and/or counting may be performed by translocating the tag/second binding member through a nanopore. Devices and systems that are programmed to carry out the disclosed methods are also provided. Also provided herein are instruments that are programmed to operate a cartridge that includes an array of electrodes for actuating a droplet and further includes an electrochemical species sensing region. The instrument may be used to analyse a sample in a cartridge that includes an array of electrodes for actuating a droplet and further includes a nanopore layer for detecting translocation of a tag/second binding member through nanopore. An instrument configured to operate a first cartridge that includes an array of electrodes for actuating a droplet and further includes an electrochemical species sensing region and a second cartridge that includes an array of electrodes for actuating a droplet and further includes a nanopore layer for detecting translocation of a tag/second binding member through nanopore is disclosed. An instrument configured to operate a cartridge that includes an array of electrodes for actuating a droplet, an electrochemical species sensing region, and a nanopore layer for detecting translocation of a tag/second binding member through nanopore is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/726,298, filed on Oct. 5, 2017, issued as U.S. Pat. No. 11,016,053,and claims priority to U.S. Provisional Application Ser. No. 62/404,722,filed on Oct. 5, 2016, and U.S. Provisional Application Ser. No.62/424,996, filed on Nov. 21, 2016, the disclosures of whichapplications are herein incorporated by reference.

TECHNICAL FIELD

This disclosure relates to methods, devices, and systems for analyteanalysis using an analyte detection device, e.g., operably coupled witha microfluidic device.

BACKGROUND

Methods and devices that can accurately analyze analyte(s) of interestin a sample are essential for diagnostics, prognostics, environmentalassessment, food safety, detection of chemical or biological warfareagents and the like. Such methods and devices not only need to beaccurate, precise and sensitive but are also advantageous when a minutesample is to be analyzed quickly and with minimal instrumentation. Assuch, there in an interest in methods and devices with improved sampleanalysis capabilities.

SUMMARY

Embodiments of the present disclosure relate to methods, systems, anddevices for analysis of analyte(s) in a sample. In certain embodiments,the sample may be a biological sample.

The method for analysis of an analyte in a sample may involve contactingthe sample with a first binding member, where the first binding memberis immobilized on a solid support and where the first binding memberspecifically binds to the analyte; contacting the solid support with asecond binding member, where the second binding member specificallybinds to the analyte and wherein the second binding member includes acleavable tag attached thereto; removing second binding member not boundto the analyte bound to the first binding member; cleaving the tagattached to the second binding member that is bound to the analyte boundto the first binding member; translocating the cleaved tag through oracross a nanopore in a layer; determining the number of tagstranslocating through the layer; determining concentration of theanalyte in the sample based on the number of tags translocating throughthe layer. In certain embodiments, the concentration of the analyte maybe determined by counting the number of tags translocating through thelayer per unit time. In other embodiments, the concentration of theanalyte may be determined by determining the time at which the number oftags translocating through the layer reaches a threshold or by setting aperiod of time and counting cumulative number of counts in the setperiod of time.

In another embodiment, the method may include combining the samplecontaining the target analyte with a known amount of the target analyteor a competitor molecule, where the target analyte (combined with thesample) or the competitor molecule are attached to a tag via a cleavablelinker to produce a tagged analyte or tagged competitor molecule,respectively, and the tagged analyte or tagged competitor moleculecompete with the target analyte for binding to a first binding member.The method may further include contacting the combined sample with thefirst binding member, where the first binding member is immobilized on asolid support and where the first binding member specifically binds tothe target analyte (and to the tagged analyte or tagged competitormolecule); contacting the solid support with buffer for an optionalwashing step; cleaving the tag attached to the tagged analyte or taggedcompetitor that is bound to the first binding member immobilized on thesolid support; translocating the cleaved tag through or across ananopore in a layer; determining the number of tags translocatingthrough the layer; determining concentration of the analyte in thesample based on the number of tags translocating through the layer. Incertain embodiments, the concentration of the analyte may be determinedby counting the number of tags translocating through the layer per unittime. In other embodiments, the concentration of the analyte may bedetermined by determining the time at which the number of tagstranslocating through the layer reaches a threshold or by setting aperiod of time and counting cumulative number of counts in the setperiod of time. In this embodiment, the number of tags translocatedthrough the nanopore or the time at which the number of tagstranslocating through the layer reaches a threshold may be inverselycorrelated to the concentration of the analyte in the sample. Forexample, the lower count or the longer the time period for reaching athreshold, the higher the concentration of the target analyte in thesample.

In one aspect, the present invention relates to a method for measuringor detecting an analyte present in a biological sample. The methodcomprising contacting the sample with a first binding member, whereinthe first binding member is immobilized on a solid support and whereinthe first binding member specifically binds to the analyte; contactingthe analyte with a second binding member, wherein the second bindingmember specifically binds to the analyte and wherein the second bindingmember comprises a cleavable tag attached thereto; removing secondbinding member not bound to the analyte bound to the first bindingmember; cleaving the tag attached to the second binding member that isbound to the analyte bound to the first binding member; translocatingthe cleaved tag through or across one or more nanopores in a layer; andassessing the tag translocating through the layer, wherein measuring thenumber of tags translocating through the layer measures the amount ofanalyte present in the sample, or wherein detecting tags translocatingthrough the layer detects that the analyte is present in the sample. Insome embodiments, measuring the tags translocating through the layer isassessed, wherein the number of tags translocating through the layermeasures the amount of analyte present in the sample. In someembodiments, detecting the tags translocating through the layer isassessed, wherein detecting tags translocating through the layer detectsthat the analyte is present in the sample.

In one aspect, the present invention relates to a method for measuringor detecting an analyte present in a biological sample. The methodcomprising contacting the sample with a first binding member, whereinthe first binding member is immobilized on a solid support and whereinthe first binding member specifically binds to the analyte; contactingthe analyte with a second binding member, wherein the second bindingmember specifically binds to the analyte and wherein the second bindingmember comprises an aptamer; removing aptamer not bound to the analytebound to the solid substrate; dissociating the aptamer bound to theanalyte and translocating the dissociated aptamer through or across oneor more nanopores in a layer; and assessing the aptamer translocatingthrough the layer, wherein measuring the number of aptamerstranslocating through the layer measures the amount of analyte presentin the sample, or wherein detecting aptamers translocating through thelayer detects that the analyte is present in the sample. In someembodiments, measuring the aptamers translocating through the layer isassessed, wherein the number of aptamers translocating through the layermeasures the amount of analyte present in the sample. In someembodiments, detecting the aptamers translocating through the layer isassessed, wherein detecting tags translocating through the layer detectsthat the analyte is present in the sample.

In one aspect, the present invention relates to an integrated digitalmicrofluidics nanopore device comprising a bottom substrate, comprisingan array of electrodes; a top substrate spaced apart from the bottomsubstrate; and a nanopore layer disposed in between the bottom and topsubstrates. The device includes a proximal portion and a distal portionand the nanopore layer is disposed in the distal portion. The array ofelectrodes in the proximal portion is configured to generate a droplet.The array of electrodes are configured to position the droplet acrossthe nanopore layer such that the droplet is split by the nanopore layerinto a first portion and a second portion, wherein at least twoelectrodes of the array of electrodes are positioned across the nanoporelayer, where the two electrodes form an anode and a cathode and operateto drive current through a nanopore in the nanopore layer when a liquiddroplet is positioned across the nanopore layer.

In one aspect, the present invention relates to an integrated digitalmicrofluidics nanopore device comprising a bottom substrate, comprisingan array of electrodes; a top substrate spaced apart from the bottomsubstrate and comprising an electrode; and a nanopore layer disposed inbetween the bottom and top substrates. The device includes a proximalportion and a distal portion and the nanopore layer is disposed in thedistal portion. The array of electrodes and the electrode in theproximal portion are configured to generate a droplet. The array ofelectrodes and the electrode are configured to position the dropletacross the nanopore layer such that the nanopore layer splits thedroplet into a first portion and a second portion, wherein at least oneelectrode of the array of electrodes is in contact with the firstportion of a droplet positioned across the nanopore layer and theelectrode in the top substrate is positioned to contact the secondportion of the droplet positioned across the nanopore layer, where thetwo electrodes form an anode and a cathode and operate to drive currentthrough a nanopore in the nanopore layer when a liquid droplet ispositioned across the nanopore layer.

In one aspect, the present invention relates to a method for measuringor detecting an analyte present in a biological sample. The methodcomprising contacting the sample with a binding member, wherein thebinding member is immobilized on a solid support and wherein the bindingmember specifically binds to the analyte; contacting the sample, whichmay contain analyte bound to the binding member, with a labeled analyte,wherein the labeled analyte is labeled with a cleavable tag; removinglabeled analyte not bound to the binding member; cleaving the tagattached to the labeled analyte that is bound to the binding member;translocating the cleaved tag through or across one or more nanopores ina layer; and assessing the tag translocating through the layer, whereinmeasuring the number of tags translocating through the layer measuresthe amount of analyte present in the sample, or detecting tagstranslocating through the layer detects that the analyte is present inthe sample. In some embodiments, measuring the tags translocatingthrough the layer is assessed, wherein the number of tags translocatingthrough the layer measures the amount of analyte present in the sample.In some embodiments, detecting the tags translocating through the layeris assessed, wherein detecting tags translocating through the layerdetects that the analyte is present in the sample.

In one aspect, the present invention relates to a method for measuringor detecting an analyte present in a biological sample. The methodcomprising contacting the sample with a binding member, wherein bindingmember is immobilized on a solid support and wherein binding memberspecifically binds to the analyte; contacting the sample, which maycontain analyte bound to the binding member, with a labeled analyte,wherein the labeled analyte comprises an aptamer; removing labeledanalyte not bound to the binding member; dissociating the aptamer boundto the labeled analyte that is bound to the binding member andtranslocating the dissociated aptamer through or across one or morenanopores in a layer; and assessing the aptamer translocating throughthe layer, wherein measuring the number of aptamers translocatingthrough the layer measures the amount of analyte present in the sample,or detecting aptamers translocating through the layer detects that theanalyte is present in the sample. In some embodiments, measuring theaptamers translocating through the layer is assessed, wherein the numberof aptamers translocating through the layer measures the amount ofanalyte present in the sample. In some embodiments, detecting theaptamers translocating through the layer is assessed, wherein detectingtags translocating through the layer detects that the analyte is presentin the sample.

In one aspect, the present invention relates to a method for measuringor detecting an analyte present in a biological sample. The methodcomprising contacting the sample with a binding member, wherein thebinding member specifically binds to the analyte, and the binding memberis labeled with a cleavable tag; contacting the sample, which maycontain analyte bound to the binding member, with an immobilizedanalyte, wherein the immobilized analyte is immobilized on a solidsupport; removing binding member not bound to the immobilized analyte;cleaving the tag attached to the binding member that is bound to theimmobilized analyte; translocating the cleaved tag through or across oneor more nanopores in a layer; and assessing the tag translocatingthrough the layer, wherein measuring the number of tags translocatingthrough the layer measures the amount of analyte present in the sample,or detecting tags translocating through the layer detects that theanalyte is present in the sample. In some embodiments, measuring thetags translocating through the layer is assessed, wherein the number oftags translocating through the layer measures the amount of analytepresent in the sample. In some embodiments, the tags translocatingthrough the layer is assessed, wherein detecting tags translocatingthrough the layer detects that the analyte is present in the sample.

In one aspect, the present invention relates to a method for measuringor detecting an analyte present in a biological sample. The methodcomprises contacting the sample with a binding member, wherein thebinding member specifically binds to the analyte, and the binding membercomprises an aptamer; contacting the sample, which may contain analytebound to the binding member, with a immobilized analyte, wherein theimmobilized analyte is immobilized on a solid support; removing bindingmember not bound to the immobilized analyte; dissociating the aptamerbound to the binding member that is bound to the immobilized analyte andtranslocating the dissociated aptamer through or across one or morenanopores in a layer; and assessing the aptamer translocating throughthe layer, wherein measuring the number of aptamers translocatingthrough the layer measures the amount of analyte present in the sample,or detecting aptamers translocating through the layer detects that theanalyte is present in the sample. In some embodiments, measuring theaptamers translocating through the layer is assessed, wherein the numberof aptamers translocating through the layer measures the amount ofanalyte present in the sample. In some embodiments, detecting theaptamers translocating through the layer is assessed, wherein detectingtags translocating through the layer detects that the analyte is presentin the sample.

In certain aspects, the tag may be an anionic polymer, a cationicpolymer, or a nanoparticle. In certain cases, the tag may include ananionic polymer, such as, an oligonucleotide polymer. In certain cases,the oligonucleotide polymer may be a deoxyribonucleic acid or aribonucleic acid. In certain cases, the oligonucleotide polymer may be aDNA aptamer or a RNA aptamer, where the aptamer does not bind to theanalyte. In exemplary cases, the tag may include a nanoparticle whichmay be a positively charged nanoparticle or a negatively chargednanoparticle.

In certain embodiments, the tag may be spherical tag, such as, adendrimer, a bead, a nanoparticle, e.g., a nanobead, and the like. Incertain embodiments, the tag may not be linear or substantially linearor elongate in shape, such as, a polymer of ribose or deoxyribose units,an oligonucleotide, and a nucleic acid, for example, DNA or RNA.

In certain cases, the first and the second binding members may beaptamers, antibodies or receptors. For example, the first binding membermay be a receptor and the second binding member may be an antibody orthe first binding member may be an antibody and the second bindingmember may be a receptor. In certain instances, the first binding membermay be a first antibody and the second binding member may be a secondantibody.

In certain instances, the tag may be negatively charged and thetranslocating may include applying a positive potential across the layerthereby translocating the tag across the layer.

In certain instances, the tag may be positively charged and thetranslocating may include applying a negative potential across the layerthereby translocating the tag across the layer.

In other embodiments, the tag may be a nucleic acid and the tag may behybridized to an oligonucleotide that includes a sequence complementaryto sequence of the tag prior to the translocating.

In another embodiment, a method for measuring an analyte present in abiological sample by using an aptamer as the second binding member isprovided. For example, the method may include contacting the sample witha first binding member, where the first binding member is immobilized ona solid support and where the first binding member specifically binds tothe analyte; contacting the analyte with a second binding member,wherein the second binding member specifically binds to the analyte andwherein the second binding member comprises an aptamer; removing aptamernot bound to the analyte bound to the solid substrate; dissociating theaptamer from the analyte that is bound to the solid substrate andtranslocating the dissociated aptamer through nanopore(s) in a layer;determining the number of aptamers translocating through the layer;measuring the analyte in the sample based on the number of aptamerstranslocating through the layer. In this embodiment, the second bindingmember is not attached to a tag as the second binding member is directlydetected by the nanopore(s).

The aptamer may be a DNA aptamer or a RNA aptamer. The first bindingmember may be an antibody. In certain instance, the analyte may be aligand and the first binding member may be a receptor.

Also disclosed herein are methods for simultaneously analyzing multipledifferent analytes in a sample, for example, the method may includeanalysis of a first and a second analyte; a first, a second, and a thirdanalyte; and so on. In certain cases, the method for analysis ofplurality of different analytes in a sample may include contacting thesample with a plurality of different first binding members, where afirst binding member of the different first binding members bindsspecifically to a first analyte of the plurality of the differentanalytes, a second binding member of the different first binding membersbinds specifically to a second analyte of the plurality of the differentanalytes, and so on. The method may further include contacting thedifferent analytes with a plurality of second binding members, where afirst binding member of the plurality of second binding members binds tothe first analyte, a second binding member of the plurality of secondbinding members binds to the second analyte, and so on. In certaininstances, each of the plurality of different second binding members mayinclude a tag that is distinct or distinguishable from each other (e.g.,each of the different second binding members has a different tag). Forexample, the first binding member of the plurality of the second bindingmembers may include a first tag, the second binding member of theplurality of the second binding members may include a second tag, and soon, where the first and second tags are distinguishable from each other.Distinguishing the tags can be done using any suitable method, e.g.,based on the nature or characteristic properties of the tags.

The method may further include removing unbound second binding members;cleaving the tags attached to the plurality of second binding membersbound to the analytes; translocating the tags through nanopores in alayer; determining the number of each of the tags translocating throughthe layer; measuring the plurality of different analytes in the samplebased on the number of each of the tags translocating through the layer.In certain embodiments, the concentration of the analyte may bedetermined by counting the number of tags translocating through thelayer per unit time. In other embodiments, the concentration of theanalyte may be determined by determining the time at which the number oftags translocating through the layer reaches a threshold. As notedherein, in certain cases, the second binding members may be a pluralityof aptamers and these aptamers are not attached to a tag as the aptamersare counted. In these embodiments, the aptamers may be dissociated fromthe analyte prior to translocating through or across a nanopore(s).

In certain cases, the different tags, such as the different aptamers,may be distinguishable from each other via nanopore force spectroscopy,optical means or electrical means or a combination thereof.

Also provided herein are kits, systems and devices for carrying out thedisclosed methods. The kits, systems and devices may be used to performanalyte analysis in an automated or a semi-automated manner andoptionally may include disposable/consumable components that areutilized for analyte analysis. Automated and semi-automated devices mayutilize microfluidics. Exemplary microfluidics include digitalmicrofluidics (DMF), surface acoustic wave (SAW) microfluidics, dropletbased microfluidic device, and the like. Exemplary microfluidics alsoinclude a fully integrated DMF and nanopore device, or a fullyintegrated SAW and nanopore device. In certain cases, the device forcarrying out the disclosed methods may be a digital microfluidics deviceused in conjunction with a nanopore device. In other embodiments, thedevice for carrying out the disclosed methods may be an integrateddigital microfluidics nanopore device. These devices may be single-usedevices or may be reusable (used multiple times for analyte analysis).The digital microfluidic and nanopore devices described herein mayprovide miniaturized, low cost analyte analysis and may be fabricatedusing low cost technologies.

Also disclosed herein is an integrated digital microfluidics nanoporedevice comprising a microfluidics module and a nanopore module; themicrofluidics module, comprising an array of electrodes spaced apartfrom a single electrode sized to overlap with at least a portion of thearray of electrodes, where the array of electrodes and the singleelectrode transport at least one droplet of fluid to a transferelectrode in the array of electrodes, wherein the transfer electrode ispositioned at an interface that operatively couples the microfluidicsmodule and the nanopore module; the nanopore module comprising a firstmicrochannel positioned on a first surface of a first substrate; asecond microchannel positioned on a first surface of a second substrate;wherein the first surface of the first substrate is in contact with thefirst surface of the second substrate thereby enclosing the firstmicrochannel and the second microchannel to provide a first capillarychannel and a second capillary channel, respectively, wherein at leastthe first capillary channel extends to the interface between themicrofluidics module and the nanopore module and is adjacent to thetransfer electrode, and is positioned to receive a fluid dropletpositioned on the transfer electrode; wherein the first capillarychannel intersects with the second capillary channel, wherein a nanoporelayer is positioned in between the first and second substrates at thelocation where the first and the second capillary channels intersect.

In certain embodiments, the array of electrodes may comprise a first anda second transfer electrodes each of which transfer electrodes areconfigured to position a fluid droplet over a surface of the transferelectrodes, wherein the first capillary channel extends to the interfacebetween the microfluidics module and the nanopore module, is adjacent tothe first transfer electrode and is positioned to receive a fluiddroplet located on the first transfer electrode and wherein the secondcapillary extends to the interface between the microfluidics module andthe nanopore module, is adjacent to the second transfer electrode and ispositioned to receive a fluid droplet located on the second transferelectrode.

In certain embodiments, the second capillary channel may not extend tothe interface and may not be connected to the electrodes of themicrofluidics module and may be connected to a vent or a reservoir onone or both ends of the second capillary. In certain cases, the secondcapillary is connected to a first reservoir at one end and a secondreservoir at the other end.

In certain embodiments, the first reservoir and/or the second reservoircomprises a fluid to be positioned across from the first capillarychannel at the intersection which fluid facilitates operation of thenanopore layer to drive current through a nanopore of the nanoporelayer. In certain embodiments, the first capillary channel and/or thesecond capillary channel varies in cross sectional width across a lengthof the capillary such that the width decreases at the intersectioncompared to the width on either sides of the intersection.

In some embodiments, the first capillary comprises a first pair ofelectrodes and the second capillary comprises a second pair ofelectrodes, wherein the first pair of electrodes is positioned in thefirst capillary channel and flank the nanopore in the nanopore layer andwherein second pair of electrodes is positioned in the second capillarychannel and flank the nanopore in the nanopore layer. The droplets maybe droplets comprising a molecule to be detected and/or counted bytransporting through the nanopore in the nanopore layer.

In certain embodiments, the fluid droplets have different compositionsand are a first droplet and a second droplet, the first dropletcomprising a molecule to be detected and/or counted by transportingacross the nanopore layer through the nanopore and the second dropletcomprising a conductive fluid lacking the molecule, where the conductivefluid facilitates transport of the molecule across the nanopore layervia the nanopore.

In certain embodiments, the first capillary channel comprises a firstelectrode positioned proximal to the nanopore layer and the secondcapillary channel comprising a second electrode positioned proximal tothe nanopore layer, wherein each of the first and second electrodes areexposed in the capillary channels such that they are in contact with afluid present in the capillary channels and wherein the first and secondelectrodes operate to drive current through a nanopore in the nanoporelayer when a liquid is positioned across the nanopore layer in the firstand second capillary channels.

In certain embodiments, the transfer electrode and the first capillarychannel are on substantially the same plane, and wherein the fluiddroplet is aligned with an opening of the first capillary channel.

In some embodiments, the transfer electrode is at a plane higher thanthe first capillary channel and wherein the device is configured with avertical port for transferring the fluid droplet down to an opening ofthe first capillary channel.

In a particular embodiment, the first surface of the first substratecomprises a first area on which the array of electrodes is disposed anda second area in which the first microchannel is formed, wherein thearray of electrodes is on a plane higher than the plane at which thefirst microchannel is formed.

In some embodiments, the second substrate comprises a notch at a sideedge located at the interface, wherein the notch is aligned over thefirst capillary channel and provides a vertical port for transport of adroplet located at the transfer electrode to the opening of the firstcapillary channel.

In some cases, the single electrode extends over the transfer electrodeand is in bi-planar configuration with the transfer electrode andwherein the single electrode and the transfer electrode operate to movethe fluid droplet to the transfer electrode.

In other cases, the single electrode extends over the transferelectrodes and is in bi-planar configuration with the transferelectrodes and wherein the single electrode and the transfer electrodesoperate to move the fluid droplets to the transfer electrodes.

In certain embodiments, the single electrode does not extend over thetransfer electrode and is not in bi-planar configuration with thetransfer electrode, wherein the fluid droplet is moved to the transferelectrode by using coplanar electrodes.

In certain embodiments, the single electrode does not extend over thetransfer electrodes and is not in bi-planar configuration with thetransfer electrodes, wherein the fluid droplets are moved to thetransfer electrodes by using coplanar electrodes.

Thus, using the devices, kits, systems and methods as described herein,analyte present in a biological sample can be measured, and a patientcan be diagnosed.

In another aspect, the present invention relates to a method ofmeasuring or detecting an analyte present in a biological samplecomprising (a) contacting the sample with a first binding member,wherein the first binding member is immobilized on a solid support andwherein the first binding member specifically binds to the analyte, (b)contacting the analyte with a second binding member, wherein the secondbinding member specifically binds to the analyte and wherein the secondbinding member comprises a cleavable tag attached thereto, (c) removingsecond binding member not bound to the analyte bound to the firstbinding member, (d) cleaving the tag attached to the second bindingmember bound to the analyte bound to the first binding member, (e)translocating the tag through one or more nanopores in a layer, and (f)assessing the tag translocating through the layer, wherein measuring thenumber of tags translocating through the layer measures the amount ofanalyte present in the sample, or wherein detecting tags translocatingthrough the layer detects that the analyte is present in the sample.

In another aspect, the present invention relates to a method ofmeasuring or detecting an analyte of interest present in a biologicalsample comprising (a) contacting the sample with a solid support, afirst specific binding member, and a second specific binding member,wherein the solid support comprises an immobilization agent, the firstspecific binding member comprises a ligand for the immobilization agentand the first specific binding member specifically binds the analyte ofinterest, the second specific binding member comprises a cleavable tag,and the second specific binding member specifically binds the analyte ofinterest, wherein a solid support/first specific binding member/analyteof interest/second specific binding member complex is formed, (b)removing second specific binding member not bound to the solidsupport/first specific binding member/analyte/second specific bindingmember complex, (c) cleaving the tag attached to the labeled analytebound to the second specific binding member in the solid support/firstspecific binding member/analyte of interest/second specific bindingmember complex, (d) translocating the tag through one or more nanoporesin a layer, and (e) assessing the tags translocating through the layer,wherein measuring the number of tags translocating through the layermeasures the amount of analyte present in the sample, or whereindetecting tags translocating through the layer detects that the analyteis present in the sample.

In another aspect, the present invention relates to a method ofmeasuring or detecting an analyte present in a biological samplecomprising (a) contacting the sample with a first binding member,wherein the first binding member is immobilized on a solid support andwherein the first binding member specifically binds to the analyte, (b)contacting the analyte with a second binding member, wherein the secondbinding member specifically binds to the analyte and wherein the secondbinding member comprises an aptamer, (c) removing aptamer not bound tothe analyte bound to the solid substrate, (d) dissociating the aptamerbound to the analyte, (e) translocating the dissociated aptamer throughone or more nanopores in a layer, and (f) assessing the aptamertranslocating through the layer, wherein measuring the number ofaptamers translocating through the layer measures the amount of analytepresent in the sample, or wherein detecting aptamers translocatingthrough the layer detects that the analyte is present in a the sample.

In one aspect, the present invention relates to an integrated digitalmicrofluidics nanopore device comprising: a first substrate, comprisingan array of electrodes; a second substrate spaced apart from the firstsubstrate; and a nanopore layer disposed between the first and secondsubstrates, wherein the array of electrodes are configured to positionthe droplet across the nanopore layer such that the droplet is split bythe nanopore layer into a first portion and a second portion, wherein atleast two electrodes of the array of electrodes are positioned acrossthe nanopore layer, where the two electrodes form an anode and a cathodeand operate to drive current through a nanopore in the nanopore layerwhen a liquid droplet is positioned across the nanopore layer.

In yet another aspect, the present invention relates to an integrateddigital microfluidics nanopore device comprising: a first substrate,comprising an array of electrodes; a second substrate spaced apart fromthe first substrate; and a nanopore layer disposed between the first andsecond substrates, wherein the array of electrodes are configured toposition a droplet across the nanopore layer such that the nanoporelayer splits the droplet into a first portion and a second portion,wherein at least one electrode of the array of electrodes is in contactwith the first portion of a droplet positioned across the nanopore layerand the electrode in the second substrate is positioned to contact thesecond portion of the droplet positioned across the nanopore layer,where the two electrodes form an anode and a cathode and operate todrive current through a nanopore in the nanopore layer when a liquiddroplet is positioned across the nanopore layer.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte present in a biological samplecomprising: (a) contacting the sample with a binding member, wherein thebinding member is immobilized on a solid support and wherein the bindingmember specifically binds to the analyte, (b) contacting the sample witha labeled analyte, wherein the labeled analyte is labeled with acleavable tag, (c) removing labeled analyte not bound to the bindingmember, (d) cleaving the tag attached to the labeled analyte bound tothe binding member, (e) translocating the tag through one or morenanopores in a layer, and (f) assessing the tags translocating throughthe layer, wherein measuring the number of tags translocating throughthe layer measures the amount of analyte present in the sample, orwherein detecting tags translocating through the layer detects that theanalyte is present in the sample.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte present in a biological sample, themethod comprising: (a) contacting the sample with a binding member,wherein binding member is immobilized on a solid support and whereinbinding member specifically binds to the analyte, (b) contacting thesample with a labeled analyte, wherein the labeled analyte comprises anaptamer; (c) removing labeled analyte not bound to the binding member,(d) dissociating the aptamer bound to the labeled analyte andtranslocating the dissociated aptamer through one or more nanopores in alayer, and (e) assessing the aptamer translocating through the layer,wherein measuring the number of aptamers translocating through the layermeasures the amount of analyte present in the sample, or whereindetecting aptamers translocating through the layer detects that theanalyte is present in the sample.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte present in a biological samplecomprising: (a) contacting the sample with a binding member, wherein thebinding member specifically binds to the analyte, and the binding memberis labeled with a cleavable tag, (b) contacting the sample with aimmobilized analyte, wherein the immobilized analyte is immobilized on asolid support, (c) removing binding member not bound to the immobilizedanalyte, (d) cleaving the tag attached to the binding member bound tothe immobilized analyte, (e) translocating the tag through one or morenanopores in a layer, and (f) assessing the tag translocating throughthe layer, wherein measuring the number of tags translocating throughthe layer measures the amount of analyte present in the sample, orwherein detecting tags translocating through the layer detects that theanalyte is present in the sample.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte present in a biological samplecomprising: (a) contacting the sample with a binding member, wherein thebinding member specifically binds to the analyte, and the binding membercomprises an aptamer, (b) contacting the sample with a immobilizedanalyte, wherein the immobilized analyte is immobilized on a solidsupport, (c) removing binding member not bound to the immobilizedanalyte, (d) dissociating the aptamer bound to the binding member boundto the immobilized analyte and translocating the dissociated aptamerthrough one or more nanopores in a layer, and (e) assessing the aptamertranslocating through the layer, wherein measuring the number ofaptamers translocating through the layer measures the amount of analytepresent in the sample, or wherein detecting aptamers translocatingthrough the layer detects that the analyte is present in the sample.

In yet another aspect, the present invention relates to an integrateddigital microfluidics nanopore device comprising a microfluidics moduleand a nanopore module; the microfluidics module comprising an array ofelectrodes, wherein the array of electrodes transports at least onedroplet of fluid to a first transfer position in the array ofelectrodes, wherein the first transfer position is at an interfacebetween the microfluidics module and the nanopore module; the nanoporemodule comprising: a first capillary channel; and a second capillarychannel; wherein at least the first capillary channel extends to theinterface and is adjacent to the first transfer position, and ispositioned to receive a fluid droplet positioned at the first transferposition; wherein the first capillary channel intersects with the secondcapillary channel, wherein a nanopore layer is positioned in between thefirst and second capillary channels at the location where the first andthe second capillary channels intersect.

In yet another aspect, the present invention relates to a method formeasuring an analyte present in a biological sample comprising: (a)contacting the sample with a first binding member, wherein the firstbinding member is immobilized on a solid support and wherein the firstbinding member specifically binds to the analyte, (b) contacting theanalyte with a second binding member, wherein the second binding memberspecifically binds to the analyte and wherein the second binding membercomprises a cleavable tag attached thereto, (c) removing second bindingmember not bound to the analyte bound to the first binding member, (d)cleaving the tag attached to the second binding member bound to theanalyte bound to the first binding member, (e) translocating the tagthrough one or more nanopores in a layer, and (f) assessing the tagtranslocating through the layer, wherein each tag translocating throughthe layer is a translocation event, wherein measuring the number oftranslocation events measures the amount of analyte present in thesample, wherein the amount of analyte present in the sample isdetermined by: i) counting the number of translocation events during aset period of time and correlating the number of translocation events toa control; ii) measuring the amount of time for a set number oftranslocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference standardcomprising a calibration curve, standard addition, or digital polymerasechain reaction, wherein the standard curve in subsection i) isdetermined by measuring the number of translocation events for controlconcentrations of analyte during a set period of time; wherein thestandard curve in subsection ii) is determined by measuring the time ittakes for a set number of translocation events to occur for controlconcentrations of analyte; and wherein the standard curve in subsectioniii) is determined by measuring the average time between translocationevents to occur for control concentrations of analyte.

In yet another aspect, the present invention relates to a method formeasuring an analyte present in a biological sample comprising: (a)contacting the sample with a first binding member, wherein the firstbinding member is immobilized on a solid support and wherein the firstbinding member specifically binds to the analyte, (b) contacting theanalyte with a second binding member, wherein the second binding memberspecifically binds to the analyte and wherein the second binding membercomprises an aptamer, (c) removing aptamer not bound to the analytebound to the solid substrate, (d) dissociating the aptamer bound to theanalyte, and (e) translocating the dissociated aptamer through one ormore nanopores in a layer; and (f) assessing the aptamer translocatingthrough the layer, wherein each aptamer translocating through the layeris a translocation event, wherein measuring the number of translocationevents measures the amount of analyte present in the sample, wherein theamount of analyte present in the sample is determined by: i) countingthe number of translocation events during a set period of time andcorrelating the number of translocation events to a control; ii)measuring the amount of time for a set number of translocation events tooccur and correlating to a control; or iii) measuring the average timebetween translocation events to occur and correlating to a control,wherein the control is a reference standard comprising a calibrationcurve, standard addition, or digital polymerase chain reaction, whereinthe standard curve in subsection i) is determined by measuring thenumber of translocation events for control concentrations of analyteduring a set period of time; wherein the standard curve in subsectionii) is determined by measuring the time it takes for a set number oftranslocation events to occur for control concentrations of analyte; andwherein the standard curve in subsection iii) is determined by measuringthe average time between translocation events to occur for controlconcentrations of analyte.

In yet another aspect, the present invention relates to method formeasuring an analyte present in a biological sample comprising: (a)contacting the sample with a binding member, wherein the binding memberis immobilized on a solid support and wherein the binding memberspecifically binds to the analyte, (b) contacting the sample with alabeled analyte, wherein the labeled analyte is labeled with a cleavabletag, (c) removing labeled analyte not bound to the binding member, (d)cleaving the tag attached to the labeled analyte bound to the bindingmember, (e) translocating the tag through one or more nanopores in alayer, and (f) assessing the tags translocating through the layer,wherein each tag translocating through the layer is a translocationevent, wherein measuring the number of translocation events measures theamount of analyte present in the sample, wherein the amount of analytepresent in the sample is determined by: i) counting the number oftranslocation events during a set period of time and correlating thenumber of translocation events to a control; ii) measuring the amount oftime for a set number of translocation events to occur and correlatingto a control; or iii) measuring the average time between translocationevents to occur and correlating to a control, wherein the control is areference standard comprising a calibration curve, standard addition, ordigital polymerase chain reaction, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

In another aspect, the present invention relates to a method formeasuring an analyte present in a biological sample comprising: (a)contacting the sample with a binding member, wherein binding member isimmobilized on a solid support and wherein binding member specificallybinds to the analyte, (b) contacting the sample with a labeled analyte,wherein the labeled analyte comprises an aptamer, (c) removing labeledanalyte not bound to the binding member, (d) dissociating the aptamerbound to the labeled analyte and translocating the dissociated aptamerthrough one or more nanopores in a layer, and (e) assessing the aptamertranslocating through the layer, wherein each aptamer translocatingthrough the layer is a translocation event, wherein measuring the numberof translocation events measures the amount of analyte present in thesample, wherein the amount of analyte present in the sample isdetermined by: i) counting the number of translocation events during aset period of time and correlating the number of translocation events toa control; ii) measuring the amount of time for a set number oftranslocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference standardcomprising a calibration curve, standard addition, or digital polymerasechain reaction, wherein the standard curve in subsection i) isdetermined by measuring the number of translocation events for controlconcentrations of analyte during a set period of time; wherein thestandard curve in subsection ii) is determined by measuring the time ittakes for a set number of translocation events to occur for controlconcentrations of analyte; and wherein the standard curve in subsectioniii) is determined by measuring the average time between translocationevents to occur for control concentrations of analyte.

In yet another aspect, the present invention relates to a method formeasuring an analyte present in a biological sample comprising: (a)contacting the sample with a binding member, wherein the binding memberspecifically binds to the analyte, and the binding member is labeledwith a cleavable tag, (b) contacting the sample with a immobilizedanalyte, wherein the immobilized analyte is immobilized on a solidsupport, (c) removing binding member not bound to the immobilizedanalyte, (d) cleaving the tag attached to the binding member bound tothe immobilized analyte, (e) translocating the tag through one or morenanopores in a layer, and (f) assessing the tag translocating throughthe layer, wherein each tag translocating through the layer is atranslocation event, wherein measuring the number of translocationevents measures the amount of analyte present in the sample, wherein theamount of analyte present in the sample is determined by: i) countingthe number of translocation events during a set period of time andcorrelating the number of translocation events to a control; ii)measuring the amount of time for a set number of translocation events tooccur and correlating to a control; or iii) measuring the average timebetween translocation events to occur and correlating to a control,wherein the control is a reference standard comprising a calibrationcurve, standard addition, or digital polymerase chain reaction, whereinthe standard curve in subsection i) is determined by measuring thenumber of translocation events for control concentrations of analyteduring a set period of time; wherein the standard curve in subsectionii) is determined by measuring the time it takes for a set number oftranslocation events to occur for control concentrations of analyte; andwherein the standard curve in subsection iii) is determined by measuringthe average time between translocation events to occur for controlconcentrations of analyte.

In yet another aspect, the present invention relates to a method formeasuring an analyte present in a biological sample comprising: (a)contacting the sample with a binding member, wherein the binding memberspecifically binds to the analyte, and the binding member comprises anaptamer, (b) contacting the sample with a immobilized analyte, whereinthe immobilized analyte is immobilized on a solid support, (c) removingbinding member not bound to the immobilized analyte, (d) dissociatingthe aptamer bound to the binding member bound to the immobilized analyteand translocating the dissociated aptamer through one or more nanoporesin a layer, and (e) assessing the aptamer translocating through thelayer, wherein each aptamer translocating through the layer is atranslocation event, wherein measuring the number of translocationevents measures the amount of analyte present in the sample, wherein theamount of analyte present in the sample is determined by: i) countingthe number of translocation events during a set period of time andcorrelating the number of translocation events to a control; ii)measuring the amount of time for a set number of translocation events tooccur and correlating to a control; or iii) measuring the average timebetween translocation events to occur and correlating to a control,wherein the control is a reference standard comprising a calibrationcurve, standard addition, or digital polymerase chain reaction, whereinthe standard curve in subsection i) is determined by measuring thenumber of translocation events for control concentrations of analyteduring a set period of time; wherein the standard curve in subsectionii) is determined by measuring the time it takes for a set number oftranslocation events to occur for control concentrations of analyte; andwherein the standard curve in subsection iii) is determined by measuringthe average time between translocation events to occur for controlconcentrations of analyte.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte present in a biological samplecomprising: (a) contacting the sample with a binding member, wherein thebinding member is immobilized on a solid support, the binding membercomprises a cleavable tag attached thereto, and the binding memberspecifically binds to the analyte, (b) removing binding member not boundto the analyte, (c) cleaving the tag attached to the binding memberbound to the analyte, (d) translocating the tag through one or morenanopores in a layer, and (e) assessing the tag translocating throughthe layer, wherein each tag translocating through the layer is atranslocation event, wherein measuring the number of translocationevents measures the amount of analyte present in the sample, wherein theamount of analyte present in the sample is determined by: i) countingthe number of translocation events during a set period of time andcorrelating the number of translocation events to a control; ii)measuring the amount of time for a set number of translocation events tooccur and correlating to a control; or iii) measuring the average timebetween translocation events to occur and correlating to a control,wherein the control is a reference comprising a calibration curve,standard addition, or digital polymerase chain reaction.

In yet another aspect, the present invention relates to an integrateddigital microfluidics nanopore-enabled device comprising: amicrofluidics module and a nanopore-enabled module; the microfluidicsmodule, comprising an array of electrodes spaced apart from a singleelectrode sized to overlap with at least a portion of the array ofelectrodes, where the array of electrodes and the single electrodetransport at least one droplet of fluid to a transfer electrode in thearray of electrodes, wherein the transfer electrode is positioned at aninterface between the microfluidics module and the nanopore-enabledmodule; the nanopore-enabled module comprising: a first microchannelpositioned on a first surface of a first substrate; a secondmicrochannel positioned on a first surface of a second substrate;wherein the first surface of the first substrate is in contact with thefirst surface of the second substrate thereby enclosing the firstmicrochannel and the second microchannel to provide a first capillarychannel and a second capillary channel, respectively, wherein at leastthe first capillary channel extends to the interface between themicrofluidics module and the nanopore-enabled module and is adjacent tothe transfer electrode, and is positioned to receive a fluid dropletpositioned on the transfer electrode; wherein the first capillarychannel intersects with the second capillary channel, wherein a layer ispositioned in between the first and second substrates at the locationwhere the first and the second capillary channels intersect, wherein thelayer is devoid of a nanopore and separates an ionic liquid present inthe first and second capillary channels, wherein the first and secondcapillary channels are in electrical connection with electrodes fordriving a voltage from the first to the second capillary channel or viceversa for creating a nanopore in the layer at the intersection of thefirst and second capillary channels.

In yet another aspect, the present invention relates to a method forgenerating a nanopore in an integrated digital microfluidicsnanopore-enabled device, the method comprising: providing an integrateddigital microfluidics nanopore-enabled device as previously describedherein; applying a voltage in the first and second capillary channels todrive current through the layer; measuring conductance across the layer;terminating application of voltage upon detection of a conductanceindicative of generation of a nanopore in the layer.

In yet another aspect, the present invention relates to an integrateddigital microfluidics nanopore device comprising: a first substratecomprising an array of electrodes; a second substrate spaced apart fromthe first substrate; an opening in the first or second substrate influid communication with a nanopore layer comprising a nanopore; and apair of electrodes configured to apply an electric field through thenanopore, wherein the array of electrodes are configured to transport atleast one droplet of fluid to the opening.

In yet another aspect, the present invention relates to a pair ofintegrated digital microfluidics nanopore devices comprising: a firstintegrated digital microfluidics nanopore device described previouslyherein, wherein the single electrode is a first single electrode, andthe capillary channel is a first capillary channel; and a secondintegrated digital microfluidics nanopore device comprising: a thirdsubstrate, comprising a fifth side and a sixth side opposite the fifthside, wherein the fifth side comprises an array of electrodes; a fourthsubstrate spaced apart from the third substrate, wherein the fourthsubstrate comprises a seventh side facing the fifth side of the thirdsubstrate and a eight side opposite the seventh side, wherein theseventh side comprises a second single electrode and wherein thenanopore layer is disposed on the eight side, wherein the fourthsubstrate comprises a second capillary channel extending from theseventh side to the eight side of the fourth substrate, wherein thenanopore layer is positioned over an opening of the capillary channel,wherein the nanopore layer is interposed between the second substrateand the fourth substrate such that the nanopore provides anelectroosmotic conduit between the first capillary channel and thesecond capillary channel, wherein the pair of detection electrodescomprises a second detection electrode that is the second singleelectrode.

In yet another aspect, the present invention relates to an integrateddigital microfluidics nanopore-enabled device comprising: a firstsubstrate, comprising a first side and a second side opposite the firstside, wherein the first side comprises an array of electrodes; a secondsubstrate spaced apart from the first substrate, wherein the secondsubstrate comprises a third side facing the first side of the firstsubstrate and a fourth side opposite the third side; a nanopore-enabledlayer devoid of a nanopore and disposed on an external side of thedevice, wherein the external side is selected from the second side orthe fourth side, wherein one of the first or second substratescomprising the external side comprises a capillary channel extendingfrom the first side to the second side of the first substrate, or thethird side to the fourth side of the second substrate, wherein thenanopore-enabled layer is positioned over an opening of the capillarychannel; and a pair of electrodes configured to apply an electric fieldacross the nanopore-enabled layer, wherein the array of electrodes areconfigured to transport at least one droplet of fluid to the capillarychannel.

In yet another aspect, the present invention relates to a method forgenerating a nanopore in an integrated digital microfluidicsnanopore-enabled device comprising: providing an integrated digitalmicrofluidics nanopore-enabled device described previously herein;submerging both sides of the nanopore-enabled layer in an ionic liquidsuch that the ionic liquid on each side of the layer is in electricalcontact with either one of the pair of detection electrodes; applying avoltage between the pair of detection electrodes to drive currentthrough the layer; measuring conductance across the layer; terminatingapplication of voltage upon detection of a conductance indicative ofgeneration of a nanopore in the layer.

In another aspect, the present invention relates to a compositioncomprising a binding member, a tag and a spacer.

In yet another aspect, the present invention relates to an integrateddigital microfluidics nanopore device comprising: a first substrate,comprising an array of electrodes; a second substrate spaced apart fromthe first substrate; and a nanopore layer having a first surface and asecond surface disposed between the first and second substrates, whereinthe array of electrodes are configured to position a first droplet atthe first surface of the nanopore layer, wherein at least two electrodesof the array of electrodes are positioned across the nanopore layer,where the two electrodes form an anode and a cathode and operate todrive current through a nanopore in the nanopore layer when a liquiddroplet is at the first surface of the nanopore layer.

In yet another aspect, the present invention relates to an integrateddigital microfluidics nanopore device comprising a microfluidics moduleand a nanopore module; the microfluidics module comprising an array ofelectrodes, where the array of electrodes transport at least one dropletof fluid to a transfer position in the array of electrodes, wherein thetransfer position is at an interface between the microfluidics moduleand the nanopore module; the nanopore module comprising: a firstcapillary channel extending from the transfer position to a nanoporelayer.

In yet another aspect, the present invention relates to an integrateddigital microfluidics nanopore device comprising: a first substrate,comprising an array of electrodes; a second substrate spaced apart fromthe first substrate; a first nanopore layer having one or more nanoporestherein; a second nanopore layer having one or more nanopores therein;and at least two electrodes for creating an electric field to drive tagsthrough a nanopore in the first and second nanopore layers.

In yet another aspect, the present invention relates to a kit comprisingany of the aforementioned devices for use in any of the aforementionedmethods.

In yet another aspect, the present invention relates to a method ofusing any of the aforementioned devices for measuring or detecting ananalyte present in a biological sample or for diagnosing a patient orscreening a blood supply.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte of interest present in a biologicalsample comprising (a) contacting the sample with a solid support, abinding member, and a labeled analyte that is labeled with a cleavabletag, wherein the solid support comprises an immobilization agent, thebinding member comprises a ligand for the immobilization agent, and thebinding member specifically binds the analyte of interest to form eithera solid support/binding member/analyte of interest complex or a solidsupport/binding member/labeled analyte complex; (b) removing labeledanalyte not bound to the binding member in the solid support/bindingmember/labeled analyte complex; (c) cleaving the tag attached to thelabeled analyte bound to the binding member in the solid support/bindingmember/labeled analyte complex; (d) translocating the tag through one ormore nanopores in a layer; and (e) assessing the tags translocatingthrough the layer, wherein measuring the number of tags translocatingthrough the layer measures the amount of analyte present in the sample,or wherein detecting tags translocating through the layer detects thatthe analyte is present in the sample.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte of interest present in a biologicalsample comprising (a) contacting the sample with a solid support, abinding member, and exogenous analyte, wherein the solid supportcomprises an immobilization agent, the exogenous analyte comprises aligand for the immobilization agent and binds the solid support so as toform a solid support/immobilized analyte complex, and the binding membercomprises a cleavable tag and specifically binds the analyte of interestto form either a solid support/analyte of interest/binding membercomplex or a solid support/immobilized analyte/binding member complex;(b) removing binding member not bound in either the solidsupport/immobilized analyte/binding member complex or the solidsupport/analyte of interest/binding member complex; (c) cleaving the tagattached to the binding member in the solid support/immobilizedanalyte/binding member complex; (d) translocating the tag through one ormore nanopores in a layer; and (e) assessing the tags translocatingthrough the layer, wherein measuring the number of tags translocatingthrough the layer measures the amount of analyte present in the sample,or wherein detecting tags translocating through the layer detects thatthe analyte is present in the sample.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte of interest present in a biologicalsample, the method comprising (a) contacting the sample with a solidsupport, a binding member, and a labeled analyte that is labeled with acleavable tag, wherein the solid support comprises an immobilizationagent, the binding member comprises a ligand for the immobilizationagent, and the binding member specifically binds the analyte of interestso as to form either a solid support/binding member/analyte of interestcomplex or a solid support/binding member/labeled analyte complex; (b)removing labeled analyte not bound to the binding member in the solidsupport/binding member/labeled analyte complex; (c) cleaving the tagattached to the labeled analyte bound to the binding member in the solidsupport/binding member/labeled analyte complex; (d) translocating thetag through one or more nanopores in a layer; and (e) assessing the tagstranslocating through the layer, wherein measuring the number of tagstranslocating through the layer measures the amount of analyte presentin the sample, or wherein detecting tags translocating through the layerdetects that the analyte is present in the sample.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte of interest present in a biologicalsample, the method comprising (a) contacting the sample with a solidsupport, a binding member, and exogenous analyte, wherein the solidsupport comprises an immobilization agent, the exogenous analytecomprises a ligand for the immobilization agent and binds the solidsupport so as to form a solid support/immobilized analyte complex, andthe binding member comprises a cleavable tag and specifically binds theanalyte of interest so as to form either a solid support/analyte ofinterest/binding member complex or a solid support/immobilizedanalyte/binding member complex; (b) removing binding member not bound ineither the solid support/immobilized analyte/binding member complex orthe solid support/analyte of interest/binding member complex; (c)cleaving the tag attached to the binding member in the solidsupport/immobilized analyte/binding member complex; (d) translocatingthe tag through one or more nanopores in a layer; and (e) assessing thetags translocating through the layer, wherein measuring the number oftags translocating through the layer measures the amount of analytepresent in the sample, or wherein detecting tags translocating throughthe layer detects that the analyte is present in the sample.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte of interest present in a biologicalsample, the method comprising (a) contacting the sample with a solidsupport, a binding member, and a labeled analyte that is labeled with anaptamer, wherein the solid support comprises an immobilization agent,the binding member comprises a ligand for the immobilization agent, andthe binding member specifically binds the analyte of interest so as toform either a solid support/binding member/analyte of interest complexor a solid support/binding member/labeled analyte complex; (b) removinglabeled analyte not bound to the binding member in the solidsupport/binding member/labeled analyte complex; (c) dissociating theaptamer attached to the labeled analyte bound to the binding member inthe solid support/binding member/labeled analyte complex; (d)translocating the dissociated aptamer through one or more nanopores in alayer; and (e) assessing the aptamer translocating through the layer,wherein measuring the number of aptamers translocating through the layermeasures the amount of analyte present in the sample, or whereindetecting aptamers translocating through the layer detects that theanalyte is present in the sample.

In yet another aspect, the present invention relates to a method formeasuring or detecting an analyte of interest present in a biologicalsample, the method comprising (a) contacting the sample with a solidsupport, a binding member, and exogenous analyte, wherein the solidsupport comprises an immobilization agent, the exogenous analytecomprises a ligand for the immobilization agent and binds the solidsupport so as to form a solid support/immobilized analyte complex, andthe binding member comprises an aptamer and specifically binds theanalyte of interest so as to form either a solid support/analyte ofinterest/binding member complex or a solid support/immobilizedanalyte/binding member complex; (b) removing binding member not bound ineither the solid support/immobilized analyte/binding member complex orthe solid support/analyte of interest/binding member complex; (c)dissociating the aptamer bound to the binding member in the solidsupport/immobilized analyte/binding member complex; (d) translocatingthe tag through one or more nanopores in a layer; and (e) assessing thetags translocating through the layer, wherein measuring the number oftags translocating through the layer measures the amount of analytepresent in the sample, or wherein detecting tags translocating throughthe layer detects that the analyte is present in the sample.

Also provided herein are instruments that are programmed to operate acartridge that includes an array of electrodes for actuating a dropletand further includes an electrochemical species sensing region. Theinstrument may be used to analyse a sample in a cartridge that includesan array of electrodes for actuating a droplet and further includes ananopore layer for detecting translocation of a molecule throughnanopore. An instrument configured to operate a first cartridge thatincludes an array of electrodes for actuating a droplet and furtherincludes an electrochemical species sensing region and a secondcartridge that includes an array of electrodes for actuating a dropletand further includes a nanopore layer for detecting translocation of amolecule through nanopore is disclosed. An instrument configured tooperate a cartridge that includes an array of electrodes for actuating adroplet, an electrochemical species sensing region, and a nanopore layerfor detecting translocation of a molecule through nanopore is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of the subject matter set forth herein, both as to itsstructure and operation, may be apparent by study of the accompanyingfigures, in which like reference numerals refer to like parts. Thecomponents in the figures are not necessarily to scale, emphasis insteadbeing placed upon illustrating the principles of the subject matter.Moreover, all illustrations are intended to convey concepts, whererelative sizes, shapes and other detailed attributes may be illustratedschematically rather than literally or precisely.

FIG. 1A and FIG. 1B depict a microfluidics device 10 used in conjunctionwith a nanopore device 15.

FIG. 2A and FIG. 2B depict a schematic of a reversibly integrated devicehaving a microfluidics module 20 combined with a nanopore module 30 viaa channel 40. FIGS. 2C-2L depict schematics of exemplary integrateddevices in which a microfluidics module is fluidically connected to ananopore module. The nanopore module includes a nanopore in a layerphysically separating two microfluidic channels at a location where thetwo microfluidic channels intersect.

FIG. 3 illustrates an exemplary integrated device which includes amicrofluidics module 300 and a nanopore module 325.

FIG. 4 provides an integrated device 400 in which the digitalmicrofluidics modules includes a built-in nanopore module.

FIG. 5A shows a top view of an integrated device. FIG. 5B shows a sideview of the integrated device of FIG. 5A.

FIG. 6 depicts an exemplary device and method of the present disclosure.

FIG. 7 depicts an exemplary device and method of the present disclosure.

FIG. 8 depicts a side view of an exemplary integrated device of thepresent disclosure.

FIG. 9 depicts an exemplary system of the present disclosure.

FIG. 10 depicts a schematic of a fabrication process of a low-cost DMFchip.

FIG. 11 depicts a single flexible DMF chip fabricated according to theschematic in FIG. 10.

FIG. 12 depicts actuation of droplets in a DMF chip, according toembodiments of the present disclosure.

FIG. 13, panels A-E depict performance of an immunoassay in a DMF chip,according to embodiments of the present disclosure.

FIGS. 14A-14C depict fabrication and design of a nanopore module,according to embodiments of the present disclosure.

FIG. 15A shows a plot of leakage current measured in real-time. FIG. 15Bdepicts a current-voltage (I-V) curve for a nanopore.

FIGS. 16A-16C show filling of a capillary channel in an integratedDMF-nanopore module device, according to embodiments of the presentdisclosure.

FIG. 17 shows a schematic diagram for droplet transfer between modulesin an integrated DMF-nanopore module device, according to embodiments ofthe present disclosure.

FIG. 18 shows a schematic diagram of a nanopore module design, accordingto embodiments of the present disclosure.

FIG. 19 shows a schematic diagram of an integrated DMF-nanopore moduledevice adapted to perform droplet transfer between the modules bypassive transport, according to embodiments of the present disclosure.

FIG. 20 shows a schematic diagram of an integrated DMF-nanopore moduledevice adapted to perform droplet transfer between the modules bypassive transport, according to embodiments of the present disclosure.

FIG. 21 is a schematic diagram of a silicon microfluidic devicecontaining silicon microchannels that allow passive movement of a liquiddroplet by passive transport, according to embodiments of the presentdisclosure.

FIG. 22 is an image of a silicon microchannel of a silicon microfluidicdevice that allows passive movement of a liquid droplet by passivetransport, according to embodiments of the present disclosure.

FIG. 23A and FIG. 23B show a schematic of a fabrication method for anintegrated nanopore sensor, according to embodiments of the presentdisclosure.

FIGS. 24A-24C display the scatter plot (level duration versus level ofblockage) for plots obtained using showing translocation events through:(FIG. 24A) nanopores comprised of regular double stranded DNA (“dsDNA”);(FIG. 24B) nanopores comprised of DBCO-modified dsDNA; and (FIG. 24C)nanopores comprised of dsDNA stars.

FIG. 25 shows a schematic of the thiol-mediated chemical cleavage.

FIG. 26A and FIG. 26B show a schematic of photocleavage experimentsperformed on magnetic microparticles.

FIG. 27 shows a schematic of the reagent placement on the DMF chip.

FIG. 28 displays a bar chart of sample versus nanopore flux (DMFcleavage) in sec⁻¹.

FIG. 29 displays the means by which a threshold for digital signalcounting is determined.

FIGS. 30A-30C show current blockages over different time periods forthree standards of 94 nM (FIG. 30A), 182 nM (FIG. 30B), and 266 nM (FIG.30C).

FIG. 31 shows a dose-response curve of number of events over a fixedamount of time (5 min).

FIG. 32 shows a dose-response curve of time required for fixed number ofevents.

FIG. 33 shows a dose-response curve of events per unit time.

FIG. 34 shows a dose-response curve of events per unit time usingSeq31-SS-biotin.

FIG. 35 shows a schematic diagram of a nanopore chamber design in asilicon nanopore module, according to embodiments of the presentdisclosure.

FIG. 36 shows a table listing the physical parameters used for COMSOLelectrical field simulations in a nanopore chamber of a silicon nanoporemodule, according to embodiments of the present disclosure.

FIG. 37 is a collection of images showing simulation results for counterion concentration gradients near a nanopore in a silicon nanoporemodule, according to embodiments of the present disclosure.

FIG. 38 is a graph showing the effects of the diameter of a SiO₂ viamade over a nanopore membrane with a nanopore on the electroosmotic flowthrough the nanopore, according to embodiments of the presentdisclosure.

FIG. 39 is a graph showing the effects of the diameter of a SiO₂ viamade over a nanopore membrane with a nanopore on the conductance throughthe nanopore, according to embodiments of the present disclosure.

FIG. 40 shows a schematic diagram of an integrated DMF-nanopore moduledevice with the nanopore module positioned on one side of the DMFmodule, according to embodiments of the present disclosure.

FIG. 41 is a collection of images showing movement of liquid from a DMFmodule through a hole in a DMF module substrate by capillary force,according to embodiments of the present disclosure.

FIG. 42 is a collection of images showing an integrated DMF-nanoporemodule device with the nanopore module positioned on one side of the DMFmodule and electrodes configured for nanopore fabrication, according toembodiments of the present disclosure.

FIG. 43 is a schematic diagram of an integrated DMF-nanopore moduledevice with the nanopore module positioned on one side of the DMFmodule, according to embodiments of the present disclosure.

FIG. 44 is a schematic diagram of an integrated DMF-nanopore moduledevice with the nanopore module positioned between two DMF modules,according to embodiments of the present disclosure.

FIG. 45 is a graph showing fabrication of a nanopore in a nanoporemembrane (a transmission electron microscope (TEM) window) by applying avoltage across the nanopore membrane, and as evidenced by dielectricbreakdown, according to embodiments of the present disclosure.

FIG. 46A and FIG. 46B are a collection of graphs showing current-voltage(I-V) curves of a nanopore formed in a membrane, before and after aconditioning process, according to embodiments of the presentdisclosure.

FIG. 47 shows a scatter plot of the averages of ratios plotted betweencounting label average diameter and nanopore size to the SNR (signal tonoise ratio).

FIG. 48, panels A-F provides a schematic of an analyte detection chipaccording to one embodiment.

FIG. 49, panels A-C provides a schematic of an analyte detection chipaccording to another embodiment.

FIG. 50 provides a schematic of an analyte detection chip according toone embodiment.

FIGS. 51A and 51B illustrate side views of an exemplary analytedetection chip.

FIGS. 52A-52E illustrate cartridges comprising DMF electrodes andoptical detection chamber.

FIG. 53 illustrates a schematic of a top view of an analyte detectionchip according to another embodiment.

FIG. 54 illustrates a schematic of an alternate exemplary analytedetection chip.

FIG. 55 provides a schematic of an exemplary hematology chip.

FIGS. 56 and 57 illustrate alternate embodiments of DMF chip withmultiple detection regions.

FIG. 58, panels A and B illustrate a schematic of exemplary analytedetection devices. FIG. 58, panel C is a schematic of a cartridgecompatible with the analyte detection devices in FIG. 58, panels A andB. FIG. 58, panels D and E illustrate cartridge adapters that allowinsertion of different types of cartridges into the same slot.

FIG. 59, panels A and B depict embodiments of a cartridge (A) and ananalyte detection device (B) that is compatible with the cartridge.

FIGS. 60A and 60B illustrate exemplary analyte detection systems with aplurality of instruments for conducting a plurality of assays.

DETAILED DESCRIPTION

Embodiments of the present disclosure relate to methods, systems, anddevices for analysis of analyte(s) in a sample. In certain embodiments,the sample may be a biological sample.

1. DEFINITIONS

Before the embodiments of the present disclosure are described, it is tobe understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

“Comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” andvariants thereof, as used herein, are intended to be open-endedtransitional phrases, terms, or words that do not preclude thepossibility of additional acts or structures. The singular forms “a,”“and” and “the” include plural references unless the context clearlydictates otherwise. The present disclosure also contemplates otherembodiments “comprising,” “consisting of” and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

“Affinity” and “binding affinity” as used interchangeably herein referto the tendency or strength of binding of the binding member to theanalyte. For example, the binding affinity may be represented by theequilibrium dissociation constant (K_(D)), the dissociation rate(k_(d)), or the association rate (k_(a)).

“Analog” as used herein refers to a molecule that has a similarstructure to a molecule of interest (e.g., nucleoside analog, nucleotideanalog, sugar phosphate analog, analyte analog, etc.). An analyte analogis a molecule that is structurally similar to an analyte but for whichthe binding member has a different affinity.

“Aptamer” as used herein refers to an oligonucleotide or peptidemolecule that can bind to pre-selected targets including smallmolecules, proteins, and peptides among others with high affinity andspecificity. Aptamers may assume a variety of shapes due to theirpropensity to form helices and single-stranded loops. An oligonucleotideor nucleic acid aptamer can be a single-stranded DNA or RNA (ssDNA orssRNA) molecule. A peptide aptamer can include a short variable peptidedomain, attached at both ends to a protein scaffold.

“Bead” and “particle” are used herein interchangeably and refer to asubstantially spherical solid support.

“Component,” “components,” or “at least one component,” refer generallyto a capture antibody, a detection reagent or conjugate, a calibrator, acontrol, a sensitivity panel, a container, a buffer, a diluent, a salt,an enzyme, a co-factor for an enzyme, a detection reagent, apretreatment reagent/solution, a substrate (e.g., as a solution), a stopsolution, and the like that can be included in a kit for assay of a testsample, such as a patient urine, serum, whole blood, tissue aspirate, orplasma sample, in accordance with the methods described herein and othermethods known in the art. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Control” as used herein refers to a reference standard for an analytesuch as is known or accepted in the art, or determined empirically usingacceptable means such as are commonly employed. A “reference standard”is a standardized substance which is used as a measurement base for asimilar substance. For example, there are documented reference standardspublished in the U.S. Pharmacopeial Convention (USP-NF), Food ChemicalsCodex, and Dietary Supplements Compendium (all of which are available athttp://www.usp.org), and other well-known sources. Methods forstandardizing references are described in the literature. Alsowell-known are means for quantifying the amounts of analyte present byuse of a calibration curve for analyte or by comparison to an alternatereference standard. A standard curve can be generated using serialdilutions or solutions of known concentrations of analyte, by massspectroscopy, gravimetric methods, and by other techniques known in theart. Alternate reference standards that have been described in theliterature include standard addition (also known as the method ofstandard addition), or digital polymerase chain reaction.

“Digital microfluidics (DMF),” “digital microfluidic module (DMFmodule),” or “digital microfluidic device (DMF device)” as usedinterchangeably herein refer to a module or device that utilizes digitalor droplet-based microfluidic techniques to provide for manipulation ofdiscrete and small volumes of liquids in the form of droplets. Digitalmicrofluidics uses the principles of emulsion science to createfluid-fluid dispersion into channels (principally water-in-oilemulsion). It allows the production of monodisperse drops/bubbles orwith a very low polydispersity. Digital microfluidics is based upon themicromanipulation of discontinuous fluid droplets within areconfigurable network. Complex instructions can be programmed bycombining the basic operations of droplet formation, translocation,splitting, and merging.

Digital microfluidics operates on discrete volumes of fluids that can bemanipulated by binary electrical signals. By using discrete unit-volumedroplets, a microfluidic operation may be defined as a set of repeatedbasic operations, i.e., moving one unit of fluid over one unit ofdistance. Droplets may be formed using surface tension properties of theliquid. Actuation of a droplet is based on the presence of electrostaticforces generated by electrodes placed beneath the bottom surface onwhich the droplet is located. Different types of electrostatic forcescan be used to control the shape and motion of the droplets. Onetechnique that can be used to create the foregoing electrostatic forcesis based on dielectrophoresis which relies on the difference ofelectrical permittivities between the droplet and surrounding medium andmay utilize high-frequency AC electric fields. Another technique thatcan be used to create the foregoing electrostatic forces is based onelectrowetting, which relies on the dependence of surface tensionbetween a liquid droplet present on a surface and the surface on theelectric field applied to the surface.

“Drag-tag” refers to a mobility modifier. The drag-tag may begenetically engineered, highly repetitive polypeptides (“proteinpolymers”) that are designed to be large, water-soluble, and completelymonodisperse. Positively charged arginines may be deliberatelyintroduced at regular intervals into the amino acid sequence to increasethe hydrodynamic drag without increasing drag-tag length. Drag-tags aredescribed in U.S. Patent Publication No. 20120141997, which isincorporated herein by reference.

“Enzymatic cleavable sequence” as used herein refers to any nucleic acidsequence that can be cleaved by an enzyme. For example, the enzyme maybe a protease or an endonuclease, such as a restriction endonuclease(also called restriction enzymes). Restriction endonucleases are capableof recognizing and cleaving a DNA molecule at a specific DNA cleavagesite between predefined nucleotides. Some endonucleases, such as forexample Fokl, comprise a cleavage domain that cleaves the DNAunspecifically at a certain position regardless of the nucleotidespresent at this position. In some embodiments, the specific DNA cleavagesite and the DNA recognition site of the restriction endonuclease areidentical.

“Globular protein” refers to a water soluble protein that has a roughlyspherical shape. Examples of globular proteins include but are notlimited to ovalbumin, beta-globulin, C-reactive protein, fibrin,hemoglobin, IgG, IgM, and thrombin.

“Label” or “detectable label” as used interchangeably herein refers to atag attached to a specific binding member or analyte by a cleavablelinker.

“Nanoparticle(s)” and “nanobead(s)” are used interchangeably herein andrefer to a nanobead or nanoparticle sized to translocate through oracross a nanopore used for counting the number ofnanobeads/nanoparticles traversing through it.

“Nucleobase” or “Base” means those naturally occurring and syntheticheterocyclic moieties commonly known in the art of nucleic acid orpolynucleotide technology or peptide nucleic acid technology forgenerating polymers. Non-limiting examples of suitable nucleobasesinclude: adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil,2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). Nucleobases can be linked to other moietiesto form nucleosides, nucleotides, and nucleoside/tide analogs.

“Nucleoside” refers to a compound consisting of a purine, deazapurine,or pyrimidine nucleobase, e.g., adenine, guanine, cytosine, uracil,thymine, 7-deazaadenine, 7-deazaguanosine, that is linked to theanomeric carbon of a pentose sugar at the 1′ position, such as a ribose,2′-deoxyribose, or a 2′,3′-di-deoxyribose.

“Nucleotide’ as used herein refers to a phosphate ester of a nucleoside,e.g., a mono-, a di-, or a triphosphate ester, wherein the most commonsite of esterification is the hydroxyl group attached to the C-5position of the pentose.

“Nucleobase polymer” or “nucleobase oligomer” refers to two or morenucleobases that are connected by linkages to form an oligomer.Nucleobase polymers or oligomers include, but are not limited to, poly-and oligonucleotides (e.g., DNA and RNA polymers and oligomers),poly-and oligo-nucleotide analogs and poly- and oligonucleotide mimics,such as polyamide or peptide nucleic acids. Nucleobase polymers oroligomers can vary in size from a few nucleobases to several hundrednucleobases or to several thousand nucleobases. The nucleobase polymersor oligomers may include from about 2 to 100 nucleobases or from about8000 to 10000 nucleobases. For example, the nucleobase polymers oroligomers may have at least about 2 nucleobases, at least about 5nucleobases, at least about 10 nucleobases, at least about 20nucleobases, at least about 30 nucleobases, at least about 40nucleobases, at least about 50 nucleobases, at least about 60nucleobases, at least about 70 nucleobases, at least about 80nucleobases, at least about 90 nucleobases, at least about 100nucleobases, at least about 200 nucleobases, at least about 300nucleobases, at least about 400 nucleobases, at least about 500nucleobases, at least about 600 nucleobases, at least about 700nucleobases, at least about 800 nucleobases, at least about 900nucleobases, at least about 1000 nucleobases, at least about 2000nucleobases, at least about 3000 nucleobases, at least about 4000nucleobases, at least about 5000 nucleobases, at least about 6000nucleobases, at least about 7000 nucleobases, at least about 8000nucleobases, at least about 9000 nucleobases, or at least about 10000nucleobases.

“One or more nanopores in a layer” means that in a single membranestructure or multiple membrane structures there is either one nanopore,or there are multiple nanopores (e.g., two or more) next to each other(e.g., side by side). When one or more nanopores are present (e.g., one,two, three, four, five, six, or other number of nanopores as technicallyfeasible), optionally they are present side by side (e.g., next to eachother) or in series (e.g., one nanopore in one layer present separatefrom or stacked onto (e.g., above or on top of) another nanopore inanother layer, etc.), or in alternate structure such as would beapparent to one skilled in the art. Optionally, such nanopores areindependently addressable, e.g., by each being within its own separatecompartment (e.g., walled off from any other nanopore), or alternatelycan be addressed by an independent detection circuit.

“Polymer brush” refers to a layer of polymers attached with one end to asurface. The polymers are close together and form a layer or coatingthat forms its own environment. The brushes may be either in a solventstate, when the dangling chains are submerged into a solvent, or in amelt state, when the dangling chains completely fill up the spaceavailable. Additionally, there is a separate class of polyelectrolytebrushes, when the polymer chains themselves carry an electrostaticcharge. The brushes may be characterized by the high density of graftedchains. The limited space then leads to a strong extension of thechains, and unusual properties of the system. Brushes may be used tostabilize colloids, reduce friction between surfaces, and to providelubrication in artificial joints

“Polynucleotides” or “oligonucleotides” refer to nucleobase polymers oroligomers in which the nucleobases are connected by sugar phosphatelinkages (sugar-phosphate backbone). Exemplary poly- andoligonucleotides include polymers of T-deoxyribonucleotides (DNA) andpolymers of ribonucleotides (RNA). A polynucleotide may be composedentirely of ribonucleotides, entirely of 2′-deoxyribonucleotides orcombinations thereof. “Nucleic acid” encompasses “polynucleotide” and“oligonucleotides” and includes single stranded and double strandedpolymers of nucleotide monomers.

“Polynucleotide analog” or “oligonucleotide analog” refers to nucleobasepolymers or oligomers in which the nucleobases are connected by a sugarphosphate backbone comprising one or more sugar phosphate analogs.Typical sugar phosphate analogs include, but are not limited to, sugaralkylphosphonates, sugar phosphoramidites, sugar alkyl- or substitutedalkylphosphotriesters, sugar phosphorothioates, sugarphosphorodithioates, sugar phosphates and sugar phosphate analogs inwhich the sugar is other than 2′-deoxyribose or ribose, nucleobasepolymers having positively charged sugar-guanidyl interlinkages such asthose described in U.S. Pat. Nos. 6,013,785 and 5,696,253.

As used herein, a “pore” (alternately referred to herein as “nanopore”)or “channel” (alternately referred to herein as “nanopore” or a“nanochannel”) refers to an orifice, gap, conduit, or groove in amembrane/layer, where the pore or channel is of sufficient dimensionthat allows passage or analysis of a single molecule (e.g., a tag) atone time (e.g., one-by-one, as in a series).

“Receptor” as used herein refers to a protein-molecule that recognizesand responds to endogenous-chemical signals. When suchendogenous-chemical signals bind to a receptor, they cause some form ofcellular/tissue-response. Examples of receptors include, but are notlimited to, neural receptors, hormonal receptors, nutrient receptors,and cell surface receptors.

As used herein, “spacer” refers to a chemical moiety that extends thecleavable group from the specific binding member, or which provideslinkage between the binding member and the support, or which extends thelabel/tag from the photocleavable moiety. In some embodiments, one ormore spacers may be included at the N-terminus or C-terminus of apolypeptide or nucleotide-based tag or label in order to distanceoptimally the sequences from the specific binding member. Spacers mayinclude but are not limited to 6-aminocaproic acid, 6-aminohexanoicacid; 1,3-diamino propane; 1,3-diamino ethane; polyethylene glycol (PEG)polymer groups, short amino acid sequences, and such as polyglycinesequences, of 1 to 5 amino acids. In some embodiments, the spacer is anitrobenzyl group, dithioethylamino, 6 carbon spacer, 12 carbon spacer,or3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonate.

“Specific binding partner” or “specific binding member” as usedinterchangeably herein refers to one of two or more different moleculesthat specifically recognize the other molecule compared to substantiallyless recognition of other molecules. The one of two different moleculeshas an area on the surface or in a cavity, which specifically binds toand is thereby defined as complementary with a particular spatial andpolar organization of the other molecule. The molecules may be membersof a specific binding pair. For example, a specific binding member mayinclude, but is not limited to, a protein, such as a receptor, anenzyme, and an antibody.

As used herein, “tag” or “tag molecule” both refer to the molecule(e.g., cleaved from the second binding member or an aptamer dissociatedfrom the target analyte) that is translocated through or across ananopore and provides an indication of the level of analyte in a sample.These terms refer to a single tag molecule or a plurality of the sametag molecule. Likewise “tags”, unless specified otherwise, refers to oneor one or more tags.

“Threshold” as used herein refers to an empirically determined andsubjective cutoff level above which acquired data is considered“signal”, and below which acquired data is considered “noise”. The useof a threshold for digital signal counting is depicted in FIG. 29. Acomputer program based on CUSUM (Cumulative Sums Algorithm) is employedto process acquired data and detect events based on threshold input fromthe user. Variation between users is avoided by detection of any manyevents as possible followed by filtering the data afterwards forspecific purposes. For example, as can be seen from this figure, eventsdetected above the set threshold impact the population of events thatare counted as signal. With a “loose” threshold a lesser number ofevents will be counted as signal. With a “tight” threshold a greaternumber of events will be counted as signal. Setting the threshold asloose or tight is a subjective choice based on the desired sensitivityor specificity for an assay, and whether in a given assessment falsepositives or false negatives would be preferred. Current blockadesignatures from DNA translocations were calculated to be 1.2 nA, whichwas based on an empirical formula relating current change to thediameter of DNA and the thickness of the nanopore membrane (H. Kwok, etal., PLoS ONE, 9(3), 392880, 2014).

As used herein, reference to movement (e.g., of a nanoparticles, tag,tag molecule, or other) “through or across” a nanopore meansalternately, through, or across, in other words, from one side toanother of a nanopore, e.g., from the cis to the trans side, or viceversa.

“Tracer” as used herein refers to an analyte or analyte fragmentconjugated to a tag or label, wherein the analyte conjugated to the tagor label can effectively compete with the analyte for sites on anantibody specific for the analyte. For example, the tracer may be ananalyte or analog of the analyte, such as cyclosporine or its analogISA247, vitamin D and its analogs, sex hormones and their analogs, etc.

“Translocation event” as used herein refers to an event in which a tagtranslocates through or across (e.g., from the cis to trans side or viceversa) the layer or nanopore.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety to disclose and describe the methods and/or materials inconnection with which the publications are cited. The materials,methods, and examples disclosed herein are illustrative only and notintended to be limiting.

2. METHODS FOR ANALYTE ANALYSIS

Provided herein are methods for analyte analysis. The method may involvesingle molecule counting. In certain embodiments, a method for analyteanalysis may involve assessing an analyte present in a sample. Incertain embodiments, the assessing may be used for determining presenceof and/or concentration of an analyte in a sample. In certainembodiments, the method may also be used for determining presence ofand/or concentration of a plurality of different analytes present in asample.

Provided herein are methods for measuring or detecting an analytepresent in a biological sample. The method includes contacting thesample with a first binding member, wherein the first binding member isimmobilized on a solid support and wherein the first binding memberspecifically binds to the analyte; contacting the analyte with a secondbinding member, wherein the second binding member specifically binds tothe analyte and wherein the second binding member includes a cleavabletag attached thereto; removing second binding member not bound to theanalyte bound to the first binding member; cleaving the tag attached tothe second binding member that is bound to the analyte bound to thefirst binding member; translocating the cleaved tag through or acrossone or more nanopores in a layer; detecting or measuring tagstranslocating through the layer; and assessing the tag translocatingthrough the layer, wherein measuring the number of tags translocatingthrough the layer measures the amount of analyte present in the sample,or wherein detecting tags translocating through the layer detects thatthe analyte is present in the sample. In some embodiments, measuring thetags translocating through the layer is assessed, wherein the number oftags translocating through the layer measures the amount of analytepresent in the sample. In some embodiments, detecting the tagstranslocating through the layer is assessed, wherein detecting tagstranslocating through the layer detects that the analyte is present inthe sample.

Provided herein are methods for measuring or detecting an analytepresent in a biological sample. The method includes contacting thesample with a first binding member, wherein the first binding member isimmobilized on a solid support and wherein the first binding memberspecifically binds to the analyte; contacting the analyte with a secondbinding member, wherein the second binding member specifically binds tothe analyte and wherein the second binding member includes an aptamer;removing aptamer not bound to the analyte bound to the solid substrate;dissociating the aptamer bound to the analyte and translocating thedissociated aptamer through or across one or more nanopores in a layer;and assessing the aptamer translocating through the layer, whereinmeasuring the number of aptamers translocating through the layermeasures the amount of analyte present in the sample, or detectingaptamers translocating through the layer detects that the analyte ispresent in the sample. In some embodiments, measuring the aptamerstranslocating through the layer is assessed, wherein the number ofaptamers translocating through the layer measures the amount of analytepresent in the sample. In some embodiments, detecting the aptamerstranslocating through the layer is assessed, wherein detecting tagstranslocating through the layer detects that the analyte is present inthe sample.

In some embodiments, each tag, such as an aptamer, translocating throughthe layer is a translocation event. Measuring the number oftranslocation events measures the amount of analyte present in thesample. In some embodiments, the amount of analyte present in the samplecan be determined by counting the number of translocation events duringa set period of time and correlating the number of translocation eventsto a control. The standard curve can be determined by measuring thenumber of translocation events for control concentrations of analyteduring a set period of time. In some embodiments, the amount of analytepresent in the sample can be determined by measuring the amount of timefor a set number of translocation events to occur and correlating to acontrol. The standard curve can be determined by measuring the time ittakes for a set number of translocation events to occur for controlconcentrations of analyte. In some embodiments, the amount of analytepresent in the sample can be determined by measuring the average timebetween translocation events to occur and correlating to a control. Thestandard curve can be determined by measuring the average time betweentranslocation events to occur for control concentrations of analyte. Insome embodiments, the control can be a reference standard comprising acalibration curve, standard addition, or digital polymerase chainreaction.

In exemplary cases, the method may include contacting the sample with afirst binding member (“binding members” alternately referred to as“specific binding members,” and as described in section c) below), wherethe first binding member is immobilized on a solid support and where thefirst binding member specifically binds to the analyte; contacting theanalyte with a second binding member, which second binding memberspecifically binds to the analyte and which second binding memberincludes a cleavable tag (“tag” as defined herein and described insection d) below) attached thereto; removing second binding member notbound to the analyte bound to the first binding member; cleaving the tagattached to the second binding member that is bound to the analyte boundto the first binding member; translocating the tag through nanopores ina layer; determining the number of tags translocating through the layer;determining concentration of the analyte in the sample based on thenumber of tags translocating through the layer. In certain embodiments,the concentration of the analyte may be determined by counting thenumber of tags translocating through the layer per unit time. In otherembodiments, the concentration of the analyte may be determined bydetermining the time at which the number of tags translocating throughthe layer reaches a threshold.

The sample may be any test sample containing or suspected of containingan analyte of interest. As used herein, “analyte”, “target analyte”,“analyte of interest” are used interchangeably and refer to the analytebeing measured in the methods and devices disclosed herein. Analytes ofinterest are further described below.

“Contacting” and grammatical equivalents thereof as used herein refer toany type of combining action which brings a binding member intosufficiently close proximity with the analyte of interest in the samplesuch that a binding interaction will occur if the analyte of interestspecific for the binding member is present in the sample. Contacting maybe achieved in a variety of different ways, including combining thesample with a binding member, exposing a target analyte to a bindingmember by introducing the binding member in close proximity to theanalyte, and the like.

In certain cases, the first binding member may be immobilized on a solidsupport. As used herein, “immobilized” refers to a stable association ofthe first binding member with a surface of a solid support. By “stableassociation” is meant a physical association between two entities inwhich the mean half-life of association is one day or more, e.g., underphysiological conditions. In certain aspects, the physical associationbetween the two entities has a mean half-life of two days or more, oneweek or more, one month or more, including six months or more, e.g., 1year or more, in PBS at 4° C. According to certain embodiments, thestable association arises from a covalent bond between the two entities,a non-covalent bond between the two entities (e.g., an ionic or metallicbond), or other forms of chemical attraction, such as hydrogen bonding,Van der Waals forces, and the like.

The solid support having a surface on which the binding reagent isimmobilized may be any convenient surface in planar or non-planarconformation, such as a surface of a microfluidic chip, an interiorsurface of a chamber, an exterior surface of a bead (as defined herein),or an interior and/or exterior surface of a porous bead. For example,the first binding member may be attached covalently or non-covalently toa bead, e.g., latex, agarose, sepharose, streptavidin, tosylactivated,epoxy, polystyrene, amino bead, amine bead, carboxyl bead, or the like.In certain embodiments, the bead may be a particle, e.g., amicroparticle. In some embodiments, the microparticle may be betweenabout 0.1 nm and about 10 microns, between about 50 nm and about 5microns, between about 100 nm and about 1 micron, between about 0.1 nmand about 700 nm, between about 500 nm and about 10 microns, betweenabout 500 nm and about 5 microns, between about 500 nm and about 3microns, between about 100 nm and 700 nm, or between about 500 nm and700 nm. For example, the microparticle may be about 4-6 microns, about2-3 microns, or about 0.5-1.5 microns. Particles less than about 500 nmare sometimes considered nanoparticles. Thus, the microparticleoptionally may be a nanoparticle between about 0.1 nm and about 500 nm,between about 10 nm and about 500 nm, between about 50 nm and about 500nm, between about 100 nm and about 500 nm, about 100 nm, about 150 nm,about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm,about 450 nm, or about 500 nm.

In certain embodiments, the bead may be a magnetic bead or a magneticparticle. Magnetic beads/particles may be ferromagnetic, ferrimagnetic,paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagneticmaterials include Fe, Co, Ni, Gd, Dy, CrO₂, MnAs, MnBi, EuO, NiO/Fe.Examples of ferrimagnetic materials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (orFeO.Fe₂O₃). Beads can have a solid core portion that is magnetic and issurrounded by one or more non-magnetic layers. Alternately, the magneticportion can be a layer around a non-magnetic core. The solid support onwhich the first binding member is immobilized may be stored in dry formor in a liquid. The magnetic beads may be subjected to a magnetic fieldprior to or after contacting with the sample with a magnetic bead onwhich the first binding member is immobilized.

After the contacting step, the sample and the first binding member maybe incubated for a sufficient period of time to allow for the bindinginteraction between the binding member and analyte to occur. Inaddition, the incubating may be in a binding buffer that facilitates thespecific binding interaction. The binding affinity and/or specificity ofthe first binding member and/or the second binding member may bemanipulated or altered in the assay by varying the binding buffer. Insome embodiments, the binding affinity and/or specificity may beincreased by varying the binding buffer. In some embodiments, thebinding affinity and/or specificity may be decreased by varying thebinding buffer.

The binding affinity and/or specificity of the first binding memberand/or the second binding member may be measured using the disclosedmethods and device described below. In some embodiments, the one aliquotof sample is assayed using one set of conditions and compared to anotheraliquot of sample assayed using a different set of conditions, therebydetermining the effect of the conditions on the binding affinity and/orspecificity. For instance, changing or altering the condition can be oneor more of removing the target analyte from the sample, adding amolecule that competes with the target analyte or the ligand forbinding, and changing the pH, salt concentration, or temperature.Additionally or alternatively, a duration of time can be the variableand changing the condition may include waiting for a duration of timebefore again performing the detection methods.

In some embodiments, after the tag or aptamer passes through the pore ofa nanopore device, the device can be reconfigured to reverse themovement direction of the tag or aptamer such that the tag or aptamercan pass through the pore again and be re-measured or re-detected, forexample, in a confirmatory assay on an infectious disease assay toconfirm the measured results.

The binding buffer may include molecules standard for antigen-antibodybinding buffers such as, albumin (e.g., BSA), non-ionic detergents(Tween-20, Triton X-100), and/or protease inhibitors (e.g., PMSF). Incertain cases, the binding buffer may be added to the microfluidic chip,chamber, etc., prior to or after adding the sample. In certain cases,the first binding member may be present in a binding buffer prior tocontacting with the sample. The length of time for binding interactionbetween the binding member and analyte to occur may be determinedempirically and may depend on the binding affinity and binding aviditybetween the binding member and the analyte. In certain embodiments, thecontacting or incubating may be for a period of 5 sec to 1 hour, suchas, 10 sec-30 minutes, or 1 minute-15 minutes, or 5 minutes-10 minutes,e.g., 10 sec, 15 sec, 30 sec, 1 minute, 5 minutes, 10 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour or 2 hours. Other conditions forthe binding interaction, such as, temperature, salt concentration, mayalso be determined empirically or may be based on manufacturer'sinstructions. For example, the contacting may be carried out at roomtemperature (21° C.-28° C., e.g., 23° C.-25° C.), 37° C., or 4° C. Incertain embodiments, an optional mixing of the sample with the firstbinding member may be carried out during the contacting step.

Following complex formation between the immobilized first binding memberand the analyte, any unbound analyte may be removed from the vicinity ofthe first binding member along with the sample while the complex of thefirst binding member and the analyte may be retained due to itsassociation with the solid support. Optionally, the solid support may becontacted with a wash buffer to remove any molecules non-specificallybound to the solid support.

After the first contacting step, and the optional removal of sampleand/or optional wash steps, the complex of the first binding member andthe analyte may be contacted with a second binding member, therebyleading to the formation of a sandwich complex in which the analyte isbound by the two binding members. An optional mixing of the secondmember with the first binding member-analyte complex may be carried outduring the second contacting step. In some embodiments, immobilizationof the analyte molecules with respect to a surface may aid in removal ofany excess second binding members from the solution without concern ofdislodging the analyte molecule from the surface. In some embodiments,the second binding member may include a tag, such as a cleavable tag,attached thereto.

As noted above, the second contacting step may be carried out inconditions sufficient for binding interaction between the analyte andthe second binding member. Following the second contacting step, anyunbound second binding member may be removed, followed by an optionalwash step. Any unbound second binding member may be separated from thecomplex of the first binding member-analyte-second binding member by asuitable means such as, droplet actuation, electrophoresis,electrowetting, dielectrophoresis, electrostatic actuation, electricfield mediated, electrode mediated, capillary force, chromatography,centrifugation, or aspiration. Upon removal of any unbound secondbinding member from the vicinity of the complex of the first bindingmember-analyte-second binding member, the tag attached to the secondbinding member present in the complex of the first bindingmember-analyte-second binding member may be separated by a suitablemeans. In some embodiments, the tag is cleaved or disassociated from thecomplex which remains after removal of unbound reagents. For example,the tag may be attached to the second binding member via a cleavablelinker (“cleavable linker” as described in section f) below). Thecomplex of the first binding member-analyte-second binding member may beexposed to a cleavage agent that mediates cleavage of the cleavablelinker.

In certain embodiments, the separation of the tag from the first bindingmember-analyte-second binding member complex is carried out underconditions that do not result in disruption of the complex, resulting inrelease of only the tag from the complex. In other cases, the separationof the tag from the first binding member-analyte-second binding membercomplex is carried out under conditions that may result in disruption ofthe complex, resulting in release of the tag, as well as one or more ofthe second binding member, the analyte, the first binding member fromthe complex. In certain embodiments, the size of the nanopore used forcounting the tag may prevent the second binding member, the analyte, thefirst binding member from translocating through the nanopore. In otherembodiments, where the complex of second binding member, the analyte,the first binding member is retained on the solid support, the nanoporemay not be sized to exclude the second binding member, the analyte, andthe first binding member.

The separation step results in the generation of a free tag that can becaused to translocate through or across a nanopore or nanopore layer (asdescribed in section f) below) under the influence of an electric field.In certain cases, the cleavage step may result in separation ofsubstantially all the tag molecule(s) attached to each of the secondbinding member in the first binding member-analyte-second binding membercomplex. The number of tag molecules can be correlated to the number ofanalyte molecules in the complex which are proportional to theconcentration of the analyte in the sample. In certain embodiments, thecorrelation between the counted tag and the analyte concentration may bedirect (higher number of tag molecules relates to higher analyteconcentration). In embodiments where a tagged competitor or taggedanalyte, such as a tracer (as defined herein), is combined with thesample, which tagged competitor or tagged analyte competes with theanalyte in the sample for binding to the first binding member, thecorrelation between the counted tag and the analyte concentration may beinverse (lower number of tag molecules relates to higher analyteconcentration). The correlation between the number of tag molecules andanalyte concentration, whether direct or inverse, may be linear orlogarithmic. Thus, the number of tag molecules translocating through thenanopore may be used to determine analyte concentration in the sample.In certain embodiments, the concentration of the analyte may bedetermined by counting the number of tags translocating through thelayer per unit time. In other embodiments, the concentration of theanalyte may be determined by determining the time at which the number oftags translocating through the layer reaches a threshold. In certainembodiments, the number of tag molecules translocating through or acrossa nanopore may be determined by the frequency of current blockage at thenanopore per unit time. Signal detection is further described in sectiong) below. As described in section d) below, the tag molecule may be ananoparticle or a nanobead (“nanoparticle” and “nanobead” as definedherein).

The number of tags incorporated in the second binding member (i.e., thenumber of tags in the tag/second binding member conjugate) provides adefined stoichiometry with the analyte. In certain embodiments, a tagmay be attached to the second binding member using a procedure thatyields a consistent number of tag(s) attached to each second bindingmember. The number of tags may be optimized based on the speed ofcounting. A faster read rate may be obtained by including more tags onthe binding member as the count rate is dependent on the concentration.The number of tags may be optimized based on the stoichiometry of tagincorporation, for example 1:1 or 1:4 incorporation rate. In someembodiments, there is a 1:5 incorporation rate. For example, one secondbinding member may have 1 tag molecule, 2 tag molecules, 3 tagmolecules, 4 tag molecules, or up to 10 tag molecules attached thereto.In some embodiments, one second binding member may have 5 tag moleculesattached thereto. A number of conjugation methods for conjugating a tagto a second binding member (e.g., a peptide, a polypeptide, a nucleicacid) are known, any of which may be used to prepare tagged secondbinding members for use in the present methods and devices. For example,site specific conjugation of a tag to an analyte specific antibody maybe carried out using thiol-maleimide chemistry, amine-succinimidylchemistry, THIOBRIDGE™ technology, using antibodies with a C- orN-terminal hexahistidine tag, antibodies with an aldehyde tag,copper-free click reaction, and the like.

In some embodiments, the methods can measure the amount of analyte bydetermining the number of translocation events. In some embodiments, oneor more translocation event(s) can correspond to a binding event betweena binding member and an analyte depending on the stoichiometry of tagincorporation into the specific binding member. For example, if one tagis incorporated per binding member, then one translocation eventrepresents the binding of the binding member to the analyte; if two tagsare incorporated per binding member, then two translocation eventsrepresents the binding of the binding member to the analyte; if threetags are incorporated per binding member, then three translocationevents represents the binding of the binding member to the analyte, etc.

In another embodiment, the second binding member may be an aptamer thatspecifically binds to the analyte. In this embodiment, a tag may not beattached to the aptamer. Rather, the aptamer is counted as ittranslocates through or across a nanopore, i.e., the aptamer serves adual function of being the second binding member and being the tag. Inthese embodiments, the aptamer in the complex of first bindingmember-analyte-aptamer complex may be dissociated from the complex byany suitable method. For example, prior to translocation through oracross a nanopore, the aptamer bound to the complex of first bindingmember-analyte may be dissociated via a denaturation step. Thedenaturation step may involve exposure to a chaotropic reagent, a highsalt solution, an acidic reagent, a basic reagent, solvent, or a heatingstep. The aptamer may then be translocated through or across a nanoporeand the number of aptamer molecules translocating through or across ananopore may be used to determine concentration of the analyte in thesample.

As noted herein, the tag or aptamer may include a nucleic acid. Incertain embodiments, the counting step using a nanopore does not includedetermining the identity of the tag or the aptamer by determiningidentity of at least a portion of the nucleic acid sequence present inthe tag/aptamer. For example, the counting step may not includedetermining a sequence of the tag/aptamer. In other embodiments, thetag/aptamer may not be sequenced, however, identity of the tag/aptamermay be determined to the extent that one tag/aptamer may bedistinguished from another tag/aptamer based on a differentiable signalassociated with the tag/aptamer due its size, conformation, charge,amount of charge and the like. Identification of tag/aptamer may beuseful in methods involving simultaneous analysis of a plurality ofdifferent analytes in a sample, for example, two, three, four, or moredifferent analytes in a sample.

In certain embodiments, the simultaneous analysis of multiple analytesin a single sample may be performed by using a plurality of differentfirst and second binding members where a pair of first and secondbinding members is specific to a single analyte in the sample. In theseembodiments, the tag associated with the second binding member of afirst pair of first and second binding members specific to a singleanalyte may be distinguishable from the tag associated with the secondbinding member of a second pair of first and second binding membersspecific to a different analyte. As noted above, a first tag may bedistinguishable from second tag based on difference in dimensions,charge, etc.

In some embodiments, the concentration of an analyte in the fluid samplethat may be substantially accurately determined is less than about 5000fM (femtomolar), less than about 3000 fM, less than about 2000 fM, lessthan about 1000 fM, less than about 500 fM, less than about 300 fM, lessthan about 200 fM, less than about 100 fM, less than about 50 fM, lessthan about 25 fM, less than about 10 fM, less than about 5 fM, less thanabout 2 fM, less than about 1 fM, less than about 500 aM (attomolar),less than about 100 aM, less than about 10 aM, less than about 5 aM,less than about 1 aM, less than about 0.1 aM, less than about 500 zM(zeptomolar), less than about 100 zM, less than about 10 zM, less thanabout 5 zM, less than about 1 zM, less than about 0.1 zM, or less.

In some cases, the limit of detection (e.g., the lowest concentration ofan analyte which may be determined in solution) is about 100 fM, about50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, about 1 fM,about 500 aM (attomolar), about 100 aM, about 50 aM, about 10 aM, about5 aM, about 1 aM, about 0.1 aM, about 500 zM (zeptomolar), about 100 zM,about 50 zM, about 10 zM, about 5 zM, about 1 zM, about 0.1 zM, or less.In some embodiments, the concentration of analyte in the fluid samplethat may be substantially accurately determined is between about 5000 fMand about 0.1 fM, between about 3000 fM and about 0.1 fM, between about1000 fM and about 0.1 fM, between about 1000 fM and about 0.1 zM,between about 100 fM and about 1 zM, between about 100 aM and about 0.1zM, or less.

The upper limit of detection (e.g., the upper concentration of ananalyte which may be determined in solution) is at least about 100 fM,at least about 1000 fM, at least about 10 pM (picomolar), at least about100 pM, at least about 100 pM, at least about 10 nM (nanomolar), atleast about 100 nM, at least about 1000 nM, at least about 10 μM, atleast about 100 μM, at least about 1000 μM, at least about 10 mM, atleast about 100 mM, at least about 1000 mM, or greater.

In some cases, the presence and/or concentration of the analyte in asample may be detected rapidly, usually in less than about 1 hour, e.g.,45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, or30 seconds.

In certain embodiments, at least some steps of the methods describedherein may be carried out on a digital microfluidics device, such as thedevice described in section 3, below. In certain embodiments, themethods of the present disclosure are carried out using a digitalmicrofluidics device in conjunction with a nanopore device. For example,the digital microfluidics device and the nanopore device may be separatedevices and a droplet containing the cleaved tag(s) or the dissociatedaptamer(s) may be generated in the microfluidics device and transportedto the nanopore device. In certain embodiments, a droplet containing thecleaved tag(s) or the dissociated aptamer(s) may be aspirated from themicrofluidics device and transported to the nanopore device usingpipette operated by a user or a robot.

In certain embodiments, the methods of the present disclosure arecarried out using a device in which a digital microfluidics module isintegrated with a nanopore module, such as the device described below.In certain embodiments, the digital microfluidics module and thenanopore module may be reversibly integrated. For example, the twomodules may be combined physically to form the integrated device andwhich device could then be separated into the individual modules. Incertain embodiments, the methods of the present disclosure are carriedout using a disposable cartridge that includes a microfluidics modulewith a built-in a nanopore module. Exemplary embodiments of the devicesused for performing the methods provided herein are described further inthe next section.

In certain cases, the microfluidics device or the microfluidics moduleof the device integrated (reversibly or fully) with the nanopore modulemay include a first substrate and a second substrate arranged in aspaced apart manner, where the first substrate is separated from thesecond substrate by a gap/space, and where at least the steps ofcontacting the sample with a first binding member, contacting theanalyte with a second binding member, removing second binding member notbound to the analyte bound to the first binding member, and cleaving thetag attached to the second binding member (that remains bound to theanalyte bound to the first binding member) is carried out in thespace/gap between the first and second substrates.

Exemplary embodiments of the present method include generating a dropletof the sample and combining the droplet of the sample with a dropletcontaining the first binding member to generate a single droplet. Thefirst binding member may be immobilized on a solid substrate, such as, abead (e.g., a magnetic bead). The single droplet may be incubated for atime sufficient to allow binding of the first binding member to ananalyte present in the sample droplet. Optionally, the single dropletmay be agitated to facilitate mixing of the sample with the firstbinding member. Mixing may be achieved by moving the single droplet backand forth, moving the single droplet around over a plurality ofelectrodes, splitting a droplet and then merging the droplets, or usingSAWs, and the like. Next, the single droplet may be subjected to amagnetic force to retain the beads at a location in the device while thedroplet may be moved away and replaced with a droplet containing asecond binding member. An optional wash step may be performed, prior toadding the second binding member, by moving a droplet of wash buffer tothe location at which the beads are retained using the magnetic force.After a period of time sufficient for the second binding member to bindthe analyte bound to the first binding member, the droplet containingthe second binding member may be moved away while the beads are retainedat the first location. The beads may be washed using a droplet of washbuffer followed by contacting the beads with a droplet containing acleavage reagent to cleave the tag attached to the second bindingmember. In embodiments where the tag is attached to the second bindingmember via a photocleavable linker, the beads may be exposed to light ofthe appropriate wavelength to cleave the linker. In certain cases, thebeads may be exposed to a droplet of buffer prior to cleavage of thephotocleavable linker. Optionally, after the washing step to remove anyunbound second binding member, a droplet containing buffer may be leftcovering the beads, the magnetic force retaining the beads at the firstlocation may be removed and the buffer droplet containing the beads maybe moved to a second location at which the photocleavage may be carriedout. The droplet containing the cleaved tags may then be moved to thenanopore device or the nanopore module portion of the integrated device.In embodiments using aptamer as the second binding member, after thewashing step to remove any unbound aptamer, a droplet containing buffermay be left covering the beads, the magnetic force retaining the beadsat the first location may be removed and the buffer droplet containingthe beads may be moved to a second location at which the dissociation ofthe aptamer may be carried out. In other embodiments, after the washingstep, the beads may be exposed to a droplet of a reagent fordissociating aptamer bound to the analyte. A droplet containing thedissociated aptamer may be moved to the nanopore while the beads may beretained in place using a magnet. The droplet containing the dissociatedaptamer may be moved to the nanopore device or the nanopore moduleportion of the integrated device.

In an alternate embodiment, the first binding member may be immobilizedon a surface of the first or the second substrate at a location in thegap/space. The step of contacting a sample with the first binding membermay include moving a droplet of the sample to the location in thegap/space at which the first binding member is immobilized. Thesubsequent steps may be substantially similar to those described abovefor first binding member immobilized on magnetic beads.

After the cleaving/dissociating step, the droplet containing the cleavedtag(s)/dissociated aptamer(s) may be moved to the nanopore device or thenanopore module of the integrated device. As noted above, the droplet(s)may be moved using a liquid transfer system, such as a pipette. Incertain cases, the microfluidic module may be fluidically connected tothe nanopore module. Fluidic connection may be achieved by connectingthe microfluidics module to the nanopore module via a channel or byplacing the nanopore module within the microfluidics module, eitherreversibly or during the manufacturing process of the integrated device.Such devices are further described in the following section.

In the above embodiments, optionally, after the combining, a droplet maybe manipulated (e.g., moved back and forth, moved in a circulardirection, oscillated, split/merged, exposed to SAW, etc.) to facilitatemixing of the sample with the assay reagents, such as, the first bindingmember, second binding member, etc.

The moving of the droplets in the integrated microfluidics nanoporedevice may be carried out using electrical force (e.g., electrowetting,dielectrophoresis, electrode-mediated, opto-electrowetting,electric-field mediated, and electrostatic actuation) pressure, surfaceacoustic waves and the like. The force used for moving the droplets maybe determined based on the specifics of the device, which are describedin the following sections a) through g) below, and for the particulardevice described in section 3.

a) Multiplexing

The methods may include one or more (or alternately two or more)specific binding members to detect one or more (or alternately two ormore) target analytes in the sample in a multiplexing assay. Each of theone or more (or alternately two or more) specific binding members bindsto a different target analyte and each specific binding member islabeled with a different tag and/or aptamer. For example, a firstspecific binding member binds to a first target analyte, a secondspecific binding member binds to a second target analyte, a thirdspecific binding member binds to a third target analyte, etc. and thefirst specific binding member is labeled with a first tag and/oraptamer, the second specific binding member is labeled with a second tagand/or aptamer, the third specific binding member is labeled with athird tag and/or aptamer, etc. In some embodiments, a first conditioncauses the cleavage or release of the first tag if the first specificbinding member is labeled with a tag or the dissociation or release ofthe first aptamer if the first specific binding member is labeled withan aptamer, a second condition causes the cleavage or release of thesecond tag if the second specific binding member is labeled with a tagor the dissociation or release of the second aptamer if the secondspecific binding member is labeled with an aptamer, a third conditioncauses the cleavage or release of the third tag if the third specificbinding member is labeled with a tag or the dissociation or release ofthe third aptamer if the third specific binding member is labeled withan aptamer, etc. In some embodiments, the conditions of the sample canbe changed at various times during the assay, allowing detection of thefirst tag or aptamer, the second tag or aptamer, the third tag oraptamer, etc., thereby detecting one or more (or alternately two ormore) target analytes. In some embodiments, the one or more (oralternately two or more) cleaved tags and/or dissociated aptamers aredetected simultaneously through the pore based on the residence durationin the nanopore, magnitude of current impedance, or a combinationthereof.

b) Exemplary Target Analytes

As will be appreciated by those in the art, any analyte that can bespecifically bound by a first binding member and a second binding membermay be detected and, optionally, quantified using methods and devices ofthe present disclosure.

In some embodiments, the analyte may be a biomolecule. Non-limitingexamples of biomolecules include macromolecules such as, proteins,lipids, and carbohydrates. In certain instances, the analyte may behormones, antibodies, growth factors, cytokines, enzymes, receptors(e.g., neural, hormonal, nutrient, and cell surface receptors) or theirligands, cancer markers (e.g., PSA, TNF-alpha), markers of myocardialinfarction (e.g., troponin, creatine kinase, and the like), toxins,drugs (e.g., drugs of addiction), metabolic agents (e.g., includingvitamins), and the like. Non-limiting embodiments of protein analytesinclude peptides, polypeptides, protein fragments, protein complexes,fusion proteins, recombinant proteins, phosphoproteins, glycoproteins,lipoproteins, or the like.

In certain embodiments, the analyte may be a post-translationallymodified protein (e.g., phosphorylated, methylated, glycosylatedprotein) and the first or the second binding member may be an antibodyspecific to a post-translational modification. A modified protein may bebound to a first binding member immobilized on a solid support where thefirst binding member binds to the modified protein but not theunmodified protein. In other embodiments, the first binding member maybind to both the unmodified and the modified protein, and the secondbinding member may be specific to the post-translationally modifiedprotein.

In some embodiments, the analyte may be a cell, such as, circulatingtumor cell, pathogenic bacteria, viruses (including retroviruses,herpesviruses, adenoviruses, lentiviruses, Filoviruses (ebola),hepatitis viruses (e.g., A, B, C, D, and E); HPV, etc.; spores, etc.

A non-limiting list of analytes that may be analyzed by the methodspresented herein include Aβ42 amyloid beta-protein, fetuin-A, tau,secretogranin II, prion protein, Alpha-synuclein, tau protein,neurofilament light chain, parkin, PTEN induced putative kinase 1, DJ-1,leucine-rich repeat kinase 2, mutated ATP13A2, Apo H, ceruloplasmin,Peroxisome proliferator-activated receptor gamma coactivator-1 alpha(PGC-1a), transthyretin, Vitamin D-binding Protein, proapoptotic kinaseR (PKR) and its phosphorylated PKR (pPKR), CXCL13, IL-12p40, CXCL13,IL-8, Dkk-3 (semen), p14 endocan fragment, Serum, ACE2, autoantibody toCD25, hTERT, CAI25 (MUC 16), VEGF, sIL-2, Osteopontin, Human epididymisprotein 4 (HE4), Alpha-Fetoprotein, Albumin, albuminuria,microalbuminuria, neutrophil gelatinase-associated lipocalin (NGAL),interleukin 18 (IL-18), Kidney Injury Molecule-1 (KIM-1), Liver FattyAcid Binding Protein (L-FABP), LMP1, BARF1, IL-8, carcinoembryonicantigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and LZTS1,alpha-amylase, carcinoembryonic antigen, CA 125, IL8, thioredoxin,beta-2 microglobulin levels—monitor activity of the virus, tumornecrosis factor-alpha receptors—monitor activity of the virus, CA15-3,follicle-stimulating hormone (FSH), leutinizing hormone (LH), T-celllymphoma invasion and metastasis 1 (TIAM1), N-cadherin, EC39,amphiregulin, dUTPase, secretory gelsolin (pGSN), PSA (prostate specificantigen), thymosin (315, insulin, plasma C-peptide, glycosylatedhemoglobin (HBA1c), C-Reactive Protein (CRP), Interleukin-6 (IL-6),ARHGDIB (Rho GDP-dissociation inhibitor 2), CFL1 (Cofilin-1), PFN1(profilin-1), GSTP1 (Glutathione S-transferase P), S100A11 (ProteinS100-A11), PRDX6 (Peroxiredoxin-6), HSPE1 (10 kDa heat shock protein,mitochondrial), LYZ (Lysozyme C precursor), GPI (Glucose-6-phosphateisomerase), HIST2H2AA (Histone H2A type 2-A), GAPDH(Glyceraldehyde-3-phosphate dehydrogenase), HSPG2 (Basementmembrane-specific heparan sulfate proteoglycan core protein precursor),LGALS3BP (Galectin-3-binding protein precursor), CTSD (Cathepsin Dprecursor), APOE (Apolipoprotein E precursor), IQGAP1 (RasGTPase-activating-like protein IQGAP1), CP (Ceruloplasmin precursor),and IGLC2 (IGLC1 protein), PCDGF/GP88, EGFR, HER2, MUC4, IGF-IR,p27(kip1), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1(3-phosphoinositide dependent protein kinase-1), TSC1, TSC2, mTOR, MIG-6(ERBB receptor feedback inhibitor 1), S6K, src, KRAS, MEKmitogen-activated protein kinase 1, cMYC, TOPO II topoisomerase (DNA) IIalpha 170 kDa, FRAP1, NRG1, ESR1, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1,FOXO3A, PPP1R1B, PXN, ELA2, CTNNB1, AR, EPHB2, KLF6, ANXA7, NKX3-1,PITX2, MKI67, PHLPP, adiponectin (ADIPOQ), fibrinogen alpha chain (FGA),leptin (LEP), advanced glycosylation end product-specific receptor (AGERaka RAGE), alpha-2-HS-glycoprotein (AHSG), angiogenin (ANG), CD14molecule (CD14), ferritin (FTH1), insulin-like growth factor bindingprotein 1 (IGFBP1), interleukin 2 receptor, alpha (IL2RA), vascular celladhesion molecule 1 (VCAM1) and Von Willebrand factor (VWF),myeloperoxidase (MPO), IL1α, TNFα, perinuclear anti-neutrophilcytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilm's Tumor-1protein, Aquaporin-1, MLL3, AMBP, VDAC1, E. coli enterotoxins(heat-labile exotoxin, heat-stable enterotoxin), influenza HA antigen,tetanus toxin, diphtheria toxin, botulinum toxins, Shiga toxin,Shiga-like toxin I, Shiga-like toxin II, Clostridium difficile toxins Aand B, etc.

Exemplary targets of nucleic acid aptamers that may be measured in asample such as an environmental sample, a biological sample obtainedfrom a patient or subject in need using the subject methods and devicesinclude: drugs of abuse (e.g. cocaine), protein biomarkers (including,but not limited to, Nucleolin, nuclear factor-kB essential modulator(NEMO), CD-30, protein tyrosine kinase 7 (PTK7), vascular endothelialgrowth factor (VEGF), MUC1 glycoform, immunoglobulin μ Heavy Chains(IGHM), Immunoglobulin E, αvβ3 integrin, α-thrombin, HIV gp120, NF-κB,E2F transcription factor, HER3, Plasminogen activator inhibitor,Tenascin C,CXCL12/SDF-1, prostate specific membrane antigen (PSMA),gastric cancer cells, HGC-27); cells (including, but not limited to,non-small cell lung cancer (NSCLC), colorectal cancer cells, (DLD-1),H23 lung adenocarcinoma cells, Ramos cells, T-cell acute lymphoblasticleukemia (T-ALL) cells, CCRF-CEM, acute myeloid leukemia (AML) cells(HL60), small-cell lung cancer (SCLC) cells, NCIH69, human glioblastomacells, U118-MG, PC-3 cells, HER-2-overexpressing human breast cancercells, SK-BR-3, pancreatic cancer cell line (Mia-PaCa-2)); andinfectious agents (including, but not limited to, Mycobacteriumtuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichiacoli O157:H7, Campylobacter jejuni, Listeria monocytogenes, Pseudomonasaeruginosa, Salmonella O8, Salmonella enteritidis).

Exemplary targets of protein or peptide aptamers that may be measured ina sample obtained from a patient or subject in need using the subjectmethods and devices include, but are not limited to: HBV core capsidprotein, CDK2, E2F transcription factor, Thymidylate synthase, Ras, EB1,and Receptor for Advanced Glycated End products (RAGE). Aptamers, anduse and methods of production thereof are reviewed in e.g., Shum et al.,J Cancer Ther. 2013 4:872; Zhang et al., Curr Med Chem. 2011; 18:4185;Zhu et al., Chem Commun (Camb). 2012 48:10472; Crawford et al., BriefFunct Genomic Proteomic. 2003 2:72; Reverdatto et al., PLoS One. 20138:e65180.

c) Samples

As used herein, “sample”, “test sample”, “biological sample” refer tofluid sample containing or suspected of containing an analyte ofinterest. The sample may be derived from any suitable source. In somecases, the sample may comprise a liquid, fluent particulate solid, orfluid suspension of solid particles. In some cases, the sample may beprocessed prior to the analysis described herein. For example, thesample may be separated or purified from its source prior to analysis;however, in certain embodiments, an unprocessed sample containing theanalyte may be assayed directly. The source of the analyte molecule maybe synthetic (e.g., produced in a laboratory), the environment (e.g.,air, soil, fluid samples, e.g., water supplies, etc.), an animal, e.g.,a mammal, a plant, or any combination thereof. In a particular example,the source of an analyte is a human bodily substance (e.g., bodilyfluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus,lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lunglavage, cerebrospinal fluid, feces, tissue, organ, or the like). Tissuesmay include, but are not limited to skeletal muscle tissue, livertissue, lung tissue, kidney tissue, myocardial tissue, brain tissue,bone marrow, cervix tissue, skin, etc. The sample may be a liquid sampleor a liquid extract of a solid sample. In certain cases, the source ofthe sample may be an organ or tissue, such as a biopsy sample, which maybe solubilized by tissue disintegration/cell lysis.

A wide range of volumes of the fluid sample may be analyzed. In a fewexemplary embodiments, the sample volume may be about 0.5 nL, about 1nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL,about 10 μL, about 50 μL, about 100 μL, about 1 mL, about 5 mL, about 10mL, or the like. In some cases, the volume of the fluid sample isbetween about 0.01 μL and about 10 mL, between about 0.01 μL and about 1mL, between about 0.01 μL and about 100 μL, between about 0.1 μL andabout 10 μL, between about 1 μL and about 100 μL, between about 10 μLand about 100 μL, or between about 10 μL and about 75 μL.

In some cases, the fluid sample may be diluted prior to use in an assay.For example, in embodiments where the source of an analyte molecule is ahuman body fluid (e.g., blood, serum), the fluid may be diluted with anappropriate solvent (e.g., a buffer such as PBS buffer). A fluid samplemay be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater,prior to use.

In some cases, the sample may undergo pre-analytical processing.Pre-analytical processing may offer additional functionality such asnonspecific protein removal and/or effective yet cheaply implementablemixing functionality. General methods of pre-analytical processing mayinclude the use of electrokinetic trapping, AC electrokinetics, surfaceacoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, orother pre-concentration techniques known in the art. In some cases, thefluid sample may be concentrated prior to use in an assay. For example,in embodiments where the source of an analyte molecule is a human bodyfluid (e.g., blood, serum), the fluid may be concentrated byprecipitation, evaporation, filtration, centrifugation, or a combinationthereof. A fluid sample may be concentrated about 1-fold, about 2-fold,about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold,about 100-fold, or greater, prior to use.

In certain embodiments, the analyte is not amplified (i.e., the copynumber of the analyte is not increased) prior to the measurement of theanalyte. For example, in cases where the analyte is DNA or RNA, theanalyte is not replicated to increase copy numbers of the analyte. Incertain cases, the analyte is a protein or a small molecule.

d) Specific Binding Members

As will be appreciated by those in the art, the binding members will bedetermined by the analyte to be analyzed. Binding members for a widevariety of target molecules are known or can be readily found ordeveloped using known techniques. For example, when the target analyteis a protein, the binding members may include proteins, particularlyantibodies or fragments thereof (e.g., antigen-binding fragments (Fabs),Fab′ fragments, F(ab′)2 fragments, recombinant antibodies, chimericantibodies, single-chain Fvs (“scFv”), single chain antibodies, singledomain antibodies, such as variable heavy chain domains (“VHH”; alsoknown as “VHH fragments”) derived from animals in the Camelidae family(VHH and methods of making them are described in Gottlin et al., Journalof Biomolecular Screening, 14:77-85 (2009)), recombinant VHHsingle-domain antibodies, and V_(NAR) fragments, disulfide-linked Fvs(“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, and functionallyactive epitope-binding fragments of any of the above, full-lengthpolyclonal or monoclonal antibodies, antibody-like fragments, etc.),other proteins, such as receptor proteins, Protein A, Protein C, or thelike. In case where the analyte is a small molecule, such as, steroids,bilins, retinoids, and lipids, the first and/or the second bindingmember may be a scaffold protein (e.g., lipocalins) or a receptor. Insome cases, binding member for protein analytes may be a peptide. Forexample, when the target analyte is an enzyme, suitable binding membersmay include enzyme substrates and/or enzyme inhibitors which may be apeptide, a small molecule and the like. In some cases, when the targetanalyte is a phosphorylated species, the binding members may comprise aphosphate-binding agent. For example, the phosphate-binding agent maycomprise metal-ion affinity media such as those describe in U.S. Pat.No. 7,070,921 and U.S. Patent Application No. 20060121544.

In certain cases, at least one of the binding members may be an aptamer,such as those described in U.S. Pat. Nos. 5,270,163, 5,475,096,5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337. Nucleic acidaptamers (e.g., single-stranded DNA molecules or single-stranded RNAmolecules) may be developed for capturing virtually any target molecule.Aptamers bind target molecules in a highly specific,conformation-dependent manner, typically with very high affinity,although aptamers with lower binding affinity can be selected. Aptamersmay distinguish between target analyte molecules based on very smallstructural differences such as the presence or absence of a methyl orhydroxyl group and certain aptamers can distinguish between D- andL-enantiomers and diastereomers. Aptamers may bind small moleculartargets, including drugs, metal ions, and organic dyes, peptides,biotin, and proteins. Aptamers can retain functional activity afterbiotinylation, fluorescein labeling, and when attached to glass surfacesand microspheres.

Nucleic acid aptamers are oligonucleotides that may be single strandedoligodeoxynucleotides, oligoribonucleotides, or modifiedoligodeoxynucleotide or oligoribonucleotides. “Modified” encompassesnucleotides with a covalently modified base and/or sugar. For example,modified nucleotides include nucleotides having sugars which arecovalently attached to low molecular weight organic groups other than ahydroxyl group at the 3′ position and other than a phosphate group atthe 5′ position. Thus modified nucleotides may also include 2′substituted sugars such as 2′-O-methyl-; 2-O-alkyl; 2-O-allyl;2′-S-alkyl; 2′-S-allyl; 2′-fluoro-; 2′-halo or 2-azido-ribose,carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, andsedoheptulose. In some embodiments, the binding member comprises anucleic acid comprising a nucleotide sequence set forth in any one ofSEQ ID NOs: 1-11.

Peptide aptamers may be designed to interfere with protein interactions.Peptide aptamers may be based on a protein scaffold onto which avariable peptide loop is attached, thereby constraining the conformationof the aptamer. In some cases, the scaffold portion of the peptideaptamer is derived from Bacterial Thioredoxin A (TrxA).

When the target molecule is a carbohydrate, potentially suitable capturecomponents (as defined herein) include, for example, antibodies,lectins, and selectins. As will be appreciated by those of ordinaryskill in the art, any molecule that can specifically associate with atarget molecule of interest may potentially be used as a binding member.

For certain embodiments, suitable target analyte/binding membercomplexes can include, but are not limited to, antibodies/antigens,antigens/antibodies, receptors/ligands, ligands/receptors,proteins/nucleic acid, enzymes/substrates and/or inhibitors,carbohydrates (including glycoproteins and glycolipids)/lectins and/orselectins, proteins/proteins, proteins/small molecules, etc.

In a particular embodiment, the first binding member may be attached toa solid support via a linkage, which may comprise any moiety,functionalization, or modification of the support and/or binding memberthat facilitates the attachment of the binding member to the support.The linkage between the binding member and the support may include oneor more chemical or physical (e.g., non-specific attachment via van derWaals forces, hydrogen bonding, electrostatic interactions,hydrophobic/hydrophilic interactions; etc.) bonds and/or chemicalspacers providing such bond(s).

In certain embodiments, a solid support may also comprise a protective,blocking, or passivating layer that can eliminate or minimizenon-specific attachment of non-capture components (e.g., analytemolecules, binding members) to the binding surface during the assaywhich may lead to false positive signals during detection or to loss ofsignal. Examples of materials that may be utilized in certainembodiments to form passivating layers include, but are not limited to:polymers, such as poly(ethylene glycol), that repel the non-specificbinding of proteins; naturally occurring proteins with this property,such as serum albumin and casein; surfactants, e.g., zwitterionicsurfactants, such as sulfobetaines; naturally occurring long-chainlipids; polymer brushes, and nucleic acids, such as salmon sperm DNA.

Certain embodiments utilize binding members that are proteins orpolypeptides. As is known in the art, any number of techniques may beused to attach a polypeptide to a wide variety of solid supports. A widevariety of techniques are known to add reactive moieties to proteins,for example, the method outlined in U.S. Pat. No. 5,620,850. Further,methods for attachment of proteins to surfaces are known, for example,see Heller, Acc. Chem. Res. 23:128 (1990).

As explained herein, binding between the binding members and theanalyte, is specific, e.g., as when the binding member and the analyteare complementary parts of a binding pair. In certain embodiments, thebinding member binds specifically to the analyte. By “specifically bind”or “binding specificity,” it is meant that the binding member binds theanalyte molecule with specificity sufficient to differentiate betweenthe analyte molecule and other components or contaminants of the testsample. For example, the binding member, according to one embodiment,may be an antibody that binds specifically to an epitope on an analyte.The antibody, according to one embodiment, can be any antibody capableof binding specifically to an analyte of interest. For example,appropriate antibodies include, but are not limited to, monoclonalantibodies, bispecific antibodies, minibodies, domain antibodies (dAbs)(e.g., such as described in Holt et al. (2014) Trends in Biotechnology21:484-490), and including single domain antibodies sdAbs that arenaturally occurring, e.g., as in cartilaginous fishes and camelid, orwhich are synthetic, e.g., nanobodies, VHH, or other domain structure),synthetic antibodies (sometimes referred to as antibody mimetics),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively. As another example, the analyte molecule may be anantibody and the first binding member may be an antigen and the secondbinding member may be a secondary antibody that specifically binds tothe target antibody or the first binding member may be a secondaryantibody that specifically binds to the target antibody and the secondbinding member may be an antigen.

In some embodiments, the binding member may be chemically programmedantibodies (cpAbs) (described in Rader (2014) Trends in Biotechnology32:186-197), bispecific cpAbs, antibody-recruiting molecules (ARMs)(described in McEnaney et al. (2012) ACS Chem. Biol. 7:1139-1151),branched capture agents, such as a triligand capture agent (described inMillward et al. (2011) J. Am. Chem. Soc. 133:18280-18288), engineeredbinding proteins derived from non-antibody scaffolds, such as monobodies(derived from the tenth fibronectin type III domain of humanfibronectin), affibodies (derived from the immunoglobulin bindingprotein A), DARPins (based on Ankyrin repeat modules), anticalins(derived from the lipocalins bilin-binding protein and human lipocalin2), and cysteine knot peptides (knottins) (described in Gilbreth andKoide, (2012) Current Opinion in Structural Biology 22:1-8; Banta et al.(2013) Annu. Rev. Biomed. Eng. 15:93-113),WW domains (described in Patelet al. (2013) Protein Engineering, Design & Selection 26(4):307-314),repurposed receptor ligands, affitins (described in Behar et al. (2013)26:267-275), and/or Adhirons (described in Tiede et al. (2014) ProteinEngineering, Design & Selection 27:145-155).

According to one embodiment in which an analyte is a biological cell(e.g., mammalian, avian, reptilian, other vertebrate, insect, yeast,bacterial, cell, etc.), the binding members may be ligands havingspecific affinity for a cell surface antigen (e.g., a cell surfacereceptor). In one embodiment, the binding member may be an adhesionmolecule receptor or portion thereof, which has binding specificity fora cell adhesion molecule expressed on the surface of a target cell type.In use, the adhesion molecule receptor binds with an adhesion moleculeon the extracellular surface of the target cell, thereby immobilizing orcapturing the cell, the bound cell may then be detected by using asecond binding member that may be the same as the first binding memberor may bind to a different molecule expressed on the surface of thecell.

In some embodiments, the binding affinity between analyte molecules andbinding members should be sufficient to remain bound under theconditions of the assay, including wash steps to remove molecules orparticles that are non-specifically bound. In some cases, for example inthe detection of certain biomolecules, the binding constant of theanalyte molecule to its complementary binding member may be between atleast about 10⁴ and about 10⁶ M⁻¹, at least about 10⁵ and about 10⁹ M⁻¹,at least about 10⁷ and about 10⁹ M⁻¹, greater than about 10⁹ M⁻¹, orgreater.

e) Tag or Label

The methods described herein may include a specific binding member boundto a tag, such as a label, to analyze an analyte by impedance. Theincorporated tags or labels do not substantially interfere with theconduct of the reaction scheme. For example, the incorporated tag orlabel does not interfere with the binding constant of or the interactionbetween the analyte and its complementary binding member. The size andnumber of incorporated tags or labels may be related to the speed ofcapture and read rate. The speed of capture and read rate may beincreased by increasing the size and/or number of incorporated tags orlabels. For example, the size and number of incorporated tags or labelsmay increase the charge and increase the capture zone of the nanopore.The incorporated tag or labels do not alter the binding member kinetics,for example, antibody kinetics, or the reaction scheme. Exemplary tagsinclude polymers such as, an anionic polymer or a cationic polymer(e.g., a polypeptide with a net positive charge, such as, polyhistidineor polylysine), where the polymer is about 5-1000 residues in length; aprotein (e.g., a globular protein) which does not cross react with thebinding member and/or interfere with the assay, a dendrimer, e.g., a DNAdendrimer; and a charged nanoparticle, e.g., a nanobead. A polymer tagmay include a nucleic acid, such as, a deoxyribonucleic acid or aribonucleic acid. A polymer tag may include a nucleobase polymer. Incertain cases, the tag may be DNA or a RNA aptamer, where the aptamerdoes not bind to the analyte. In cases, where the tag is an aptamer, itmay be optionally denatured prior to the translocation through thenanopore. A polymer tag or a nanoparticle (e.g., a nanobead) may besufficiently large to generate a reproducible signal as it translocatesthrough or across a nanopore. Aptamers may be 20-220 bases in length,e.g., 20-60 bases long. The size of the nanoparticle (e.g., a nanobeador a dendrimer) may range from about 1 nm to about 950 nm in diameterfor example, 10 nm-900 nm, 20 nm-800 nm, 30 nm-700 nm, 50 nm-600 nm, 80nm-500 nm, 100 nm-500 nm, 200 nm-500 nm, 300 nm-500 nm, or 400 nm-500 nmin diameter, e.g., 10 nm, 20 nm, 30 nm, 50 nm, 100 nm, 200 nm, 300 nm,400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm. When used as a tag, apreferred size for nanoparticle is one that can pass through or across ananopore (as further described herein). In certain cases, thenanobead/nanoparticle may be made of a material that has a net negativeor positive charge or can be treated to have a net negative or positivecharge. Exemplary nanobeads/nanoparticles include those made fromorganic or inorganic polymers. Organic polymers include polymers suchas, polystyrene, carbon, polyacrylamide, etc. Inorganic polymers includesilicon or metal nanobeads/nanoparticles. In certain cases, thenanobeads/nanoparticles may not be magnetic.

In certain cases, the tag may be a single stranded DNA or RNA. Thesingle stranded DNA or RNA may be hybridized to a probe molecule priorto translocation through or across a nanopore. In certain cases, themethod may include analysis of multiple analytes in a single sample. Thesecond binding members that bind to the different analytes in a samplemay include different single stranded DNA or RNA attached thereto astags and the different single stranded DNA or RNA may be hybridized todifferent probes that further distinguish the different single strandedDNA or RNA from each other as they traverse though the nanopores. Inother embodiments, the tags attached to the different second bindingmembers may have different hairpin structures (e.g., length of thehairpin structure) that are distinguishable when the tags pass throughor across a nanopore. In yet another embodiment, the tags attached tothe different second binding members may have different lengths that aredistinguishable when the tags traverse through or across thenanopores—for example, the tags may be double stranded DNA of differentlengths (e.g., 25 bp, 50 bp, 75 bp, 100 bp, 150 bp, 200 bp, or more). Incertain cases, the tags attached to the different second binding membersmay have different lengths of polyethylene glycol (PEG) or may be DNA orRNA modified differentially with PEG.

It is noted that reference to a tag or a tag molecule encompasses asingle tag or a single tag molecule as well as multiple tags (that allmay be identical). It is further noted that the nanopore encompasses asingle nanopore as well as multiple nanopores present in a single layer,such as, a substrate, a membrane, and the like. As such, counting thenumber of tags translocating through or across a nanopore in alayer/sheet/membrane refers to counting multiple tags translocatingthrough or across one or more nanopores in a layer/sheet/membrane.Nanopores may be present in a single layer, such as a substrate or amembrane, the layer may be made of any suitable material that iselectrically insulating or has a high electrical resistance, such as alipid bilayer, a dielectric material, e.g., silicon nitride and silica,atomically thin membrane such as graphene, silicon, silicene, molybdenumdisulfide (MoS₂), etc., or a combination thereof.

The tag may be any size or shape. In some embodiments, the tag may be ananoparticle or a nanobead about 10 and 950 nm in diameter, e.g., 20-900nm, 30-800 nm, 40-700 nm, 50-600 nm, 60-500 nm, 70-400 nm, 80-300 nm,90-200 nm, 100-150 nm, 200-600 nm, 400-500 nm, 2-10 nm, 2-4 nm, or 3-4nm in diameter. The tag may be substantially spherical, for example aspherical bead or nanobead, or hemi-spherical. The tag may be a proteinabout 0.5 kDa to about 50 kDa in size, e.g., about 0.5 kDa to about 400kDa, about 0.8 kDa to about 400 kDa, about 1.0 kDa to about 400 kDa,about 1.5 kDa to about 400 kDa, about 2.0 kDa to about 400 kDa, about 5kDa to about 400 kDa, about 10 kDa to about 400 kDa, about 50 kDa toabout 400 kDa, about 100 kDa to about 400 kDa, about 150 kDa to about400 kDa, about 200 kDa to about 400 kDa, about 250 kDa to about 400 kDa,about 300 kDa to about 400 kDa, about 0.5 kDa to about 300 kDa, about0.8 kDa to about 300 kDa, about 1.0 kDa to about 300 kDa, about 1.5 kDato about 300 kDa, about 2.0 kDa to about 300 kDa, about 5 kDa to about300 kDa, about 10 kDa to about 300 kDa, about 50 kDa to about 300 kDa,about 100 kDa to about 300 kDa, about 150 kDa to about 300 kDa, about200 kDa to about 300 kDa, about 250 kDa to about 300 kDa, about 0.5 kDato about 250 kDa, about 0.8 kDa to about 250 kDa, about 1.0 kDa to about250 kDa, about 1.5 kDa to about 250 kDa, about 2.0 kDa to about 250 kDain size, about 5 kDa to about 250 kDa, about 10 kDa to about 250 kDa,about 50 kDa to about 250 kDa, about 100 kDa to about 250 kDa, about 150kDa to about 250 kDa, about 200 kDa to about 250 kDa, about 0.5 kDa toabout 200 kDa, about 0.8 kDa to about 200 kDa, about 1.0 kDa to about200 kDa, about 1.5 kDa to about 200 kDa, about 2.0 kDa to about 200 kDain size, about 5 kDa to about 200 kDa, about 10 kDa to about 200 kDa,about 50 kDa to about 200 kDa, about 100 kDa to about 200 kDa, about 150kDa to about 200 kDa, about 0.5 kDa to about 100 kDa, about 0.8 kDa toabout 100 kDa, about 1.0 kDa to about 100 kDa, about 1.5 kDa to about100 kDa, about 2.0 kDa to about 100 kDa, about 5 kDa to about 100 kDa,about 10 kDa to about 100 kDa, about 50 kDa to about 100 kDa, about 0.5kDa to about 50 kDa, about 0.8 kDa to about 50 kDa, about 1.0 kDa toabout 50 kDa, about 1.5 kDa to about 50 kDa, about 2.0 kDa to about 50kDa, about 5 kDa to about 50 kDa, about 10 kDa to about 50 kDa. about 10kDa to about 90 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about70 kDa, about 10 kDa to about 60 kDa, about 20 kDa to about 90 kDa,about 20 kDa to about 80 kDa, about 20 kDa to about 70 kDa, about 20 kDato about 60 kDa, about 40 kDa to about 90 kDa, about 40 kDa to about 80kDa, about 40 kDa to about 70 kDa, or about 40 kDa to about 60 kDa.

In certain embodiments, the tag may be a nanoparticle. As noted herein,the nanoparticle may be reversibly (e.g., cleavably) attached to thesecond binding member. In certain aspects, the nanoparticle may be ananobead of a defined diameter which may the property of the nanobeadmeasured by the nanopore layer. In certain cases, the methods, systems,and devices of the present disclosure may be used to simultaneouslyanalyze a plurality of different analytes in a sample. For such analysisa plurality of second binding members that each specifically bind to acognate analyte may be used. Each of the different second binding membermay be attached to a different sized nanobead that may be used toidentify the second binding member. For example, the different nanobeadtags may have different diameters, such as, 1 nm, 2 nm, 4 nm, 6 nm, 8nm, 10 nm, 12 nm, 14 nm, or larger, such as up to 20 nm, 30 nm, 50 nm,100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm,950 nm, or 990 nm.

In certain embodiments, the nanobeads of different diameters may alltranslocate through a nanopore layer having nanopores of a singlediameter, where the different sized nanobeads may be identified based onthe residence duration in the nanopore, magnitude of current impedance,or a combination thereof. In certain cases, a stacked nanopore layerdevice containing multiple nanopore layers, where a first layer may havenanopores of a first diameter and the second layer may have nanopores ofa second diameter may be used to detect and count the nanobeadstranslocating through or across the nanopores. The multiple nanoporelayers may be arranged in a manner such that layer with nanopores of alarger diameter is placed upstream to layer having nanopores of asmaller diameter. Exemplary stacked nanopore layers are disclosed inUS20120080361.

Exemplary nanoparticles that may be used as tags in the present methodsinclude gold nanoparticles or polystyrene nanoparticles ranging indiameter from 5 nm-950 nm.

In certain cases, the tag may be a polymer, such as, a nucleic acid. Thepresence of the tag may be determined by detecting a signalcharacteristic of the tag, such as a signal related to the size orlength of the polymer tag. The size or length of the polymer tag can bedetermined by measuring its residence time in the pore or channel, e.g.,by measuring duration of transient blockade of current.

Elements which can be part of, all of, associated with, or attached tothe tag or label include: a nanoparticle; gold particle; silverparticle; silver, copper, zinc, or other metal coating or deposit;polymer; drag-tag (as defined herein); magnetic particle; buoyantparticle; metal particle; charged moiety; dielectrophoresis tag, silicondioxide, with and without impurities (e.g., quartz, glass, etc.);poly(methylmethacrylate) (PMMA); polyimide; silicon nitride; gold;silver; quantum dot (including CdS quantum dot); carbon dot; afluorophore; a quencher; polymer; polystyrene; Janus particle;scattering particle; fluorescent particle; phosphorescent particle;sphere; cube; insulator; conductor; bar-coded or labeled particle;porous particle; solid particle; nanoshell; nanorod; microsphere;analyte such as a virus, cell, parasite and organism; nucleic acid;protein; molecular recognition element; spacer; PEG; dendrimer; chargemodifier; magnetic material; enzyme; DNA including aptamer sequence;amplifiable DNA; repeated sequence of DNA; fusion or conjugate ofdetectable elements with molecular recognition elements (e.g.,engineered binding member); anti-antibody aptamer; aptamer directed toantibody-binding protein; absorbed or adsorbed detectable compound;heme; luciferin; a phosphor; an azido, or alkyne (e.g., terminal ornon-terminal alkyne) or other click chemistry participant.

In certain embodiments, the tag may be chosen to provide a rate ofcapture that is sufficiently high to enable a rapid analysis of asample. In certain embodiments, the capture rate of the tag may be about1 event per 10 seconds, 1 event per 5 seconds, 1 event per second orhigher. In certain embodiments, linear polymer tags, such as, ribosepolymers, deoxyribose polymers, oligonucleotides, DNA, or RNA may beused. Typically for 1 nM solution of DNA, capture rates areapproximately 1 event sec⁻¹ using a solid-state nanopore (Si₃N₄), withno salt gradient, a voltage of 200-800 mV, and a salt (KCl)concentration of 1 M.

In certain cases, linear polymer tags, such as, ribose polymers,deoxyribose polymers, oligonucleotides, DNA, or RNA may not be used asthe capture rate for these tags may be too low for certain applications.Tags that are hemispherical, spherical or substantially spherical inshape rapidly translocate through the nanopores and thus shorten theassay duration may be used in applications requiring faster tagcounting. In certain cases, the size of the spherical or hemisphericaltag may be chosen based on the capture rate needed for the assay. Forexample, for a higher capture rate, spherical or hemispherical tags oflarger size may be selected. In certain cases, the tag may be sphericaltag, such as, a nanoparticle/nanobead that has a capture rate about a 10times, 30 times, 50 times, 100 times, 300 times, 500 times, or a 1000times faster than capture rate for a linear tag, such as, a DNA tag,under the same measurement conditions.

In some embodiments, the tag may be conjugated to an antibody, forexample, a CPSP antibody conjugate. In some embodiments, the tag may beconjugated to an antibody with a spacer, for example, a CPSP antibodyconjugate with a spacer. In some embodiments, the tag may be may beconjugated to an oligonucleotide and an antibody, for example, a CPSPoligonucleotide-antibody conjugate. In some embodiments, the tag may bemay be conjugated to an oligonucleotide and an antibody with a spacer,for example, a CPSP oligonucleotide-antibody conjugate with spacer. Insome embodiments, the tag may be may be conjugated to anoligonucleotide, for example, a CPSP oligonucleotide conjugate. In someembodiments, the spacer includes a nitrobenzyl group, dithioethylamino,6 carbon spacer, 12 carbon spacer, or3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonate.In some embodiments, the spacer comprises a nitrobenzyl group, and thetag is a DNA molecule. In some embodiments, the spacer isdithioethylamino and the tag is a carboxylated nanoparticle. In someembodiments, the spacer is3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonateand the tag is an oligonucleotide. In some embodiments, the spacercomprises a 6 carbon spacer or a 12 carbon spacer and the tag is biotin.

f) Cleavable Linker

The tags used in the methods described herein may be attached tospecific binding member by a generic linker. The cleavable linkerensures that the tag can be removed. The generic linker may be acleavable linker. For example, the tag may be attached to the secondbinding member via a cleavable linker. The complex of the first bindingmember-analyte-second binding member may be exposed to a cleavage agentthat mediates cleavage of the cleavable linker. The linker can becleaved by any suitable method, including exposure to acids, bases,nucleophiles, electrophiles, radicals, metals, reducing or oxidizingagents, light, temperature, enzymes etc. Suitable linkers can be adaptedfrom standard chemical blocking groups, as disclosed in Greene & Wuts,Protective Groups in Organic Synthesis, John Wiley & Sons. Furthersuitable cleavable linkers used in solid-phase synthesis are disclosedin Guillier et al. (Chem. Rev. 100:2092-2157, 2000). The linker may beacid-cleavable, base-cleavable or photocleavable. A redox reaction maybe part of the cleavage scheme. The cleavable linker may be a chargedpolymer.

The linker may be a photocleavable linker, a chemically cleavablelinker, or a thermally cleavable linker. In embodiments, the linker maybe thermal-sensitive cleavable linker. Where the linker is aphotocleavable group, the cleavage agent may be light of appropriatewavelength that disrupts or cleaves the photocleavable group. In manyembodiments, the wavelength of light used to cleave the photocleavablelinking group ranges from about 180 nm to 400 nm, e.g., from about 250nm to 400 nm, or from about 300 nm to 400 nm. It is preferable that thelight required to activate cleavage does not affect the other componentsof the analyte. Suitable linkers include those based on O-nitrobenzylcompounds and nitroveratryl compounds. Linkers based on benzoinchemistry can also be used (Lee et al., J. Org. Chem. 64:3454-3460,1999). In some embodiments, the photocleavable linker may be derivedfrom the following moiety:

Alternatively, where the cleavage linker is a chemically cleavablegroup, the cleavage agent may be a chemical agent capable of cleavingthe group. A chemically cleavable linker may be cleaved byoxidation/reduction-based cleavage, acid-catalyzed cleavage,base-catalyzed cleavage, or nucleophilic displacement. For example,where the linking group is a disulfide, thiol-mediated cleavage withdithiothreitol or betamercaptoethanol may be used to release the tag. Inyet other embodiments where the linking group is a restriction site, theagent is a catalytic agent, such as an enzyme which may be a hydrolyticenzyme, a restriction enzyme, or another enzyme that cleaves the linkinggroup. For example, the restriction enzyme may be a type I, type II,type IIS, type III and type IV restriction enzyme.

In some embodiments, the cleavage linker is an enzymatic cleavablesequence. In one aspect of any of the embodiments herein, an enzymaticcleavable sequence is a nucleic acid sequence of 2, 3, 4, 5, 6, 7, 8, 9or 10 nucleotides in length. In one embodiment, the enzymatic cleavablesequence comprises a sequence of at least 10 nucleotides. In oneembodiment, the enzymatic cleavable sequence comprises a sequence ofbetween 2 and 20 nucleotides. In one embodiment, the enzymatic cleavablesequence comprises a sequence of between 2 and 15 nucleotides. In oneembodiment, the enzymatic cleavable sequence comprises a sequence ofbetween 4 and 10 nucleotides. In one embodiment, the enzymatic cleavablesequence comprises a sequence of between 4 and 15 nucleotides.

For example, the cleavable linker may be an acridinium, ethers such assubstituted benzyl ether or derivatives thereof (e.g., benzylhydrylether, indanyl ether, etc.) that can be cleaved by acidic or mildreductive conditions (e.g., hydrogen peroxide to produce an acridone anda sulfonamide), a charged polymer generated using P-elimination, where amild base can serve to release the product, acetals, including the thioanalogs thereof, where detachment is accomplished by mild acid,particularly in the presence of a capturing carbonyl compound,photolabile linkages (e.g., O-nitrobenzoyl, 7-nitroindanyl,2-nitrobenzhydryl ethers or esters, etc.), or peptide linkers, which aresubject to enzymatic hydrolysis (e.g., enzymatic cleavable linkers),particularly where the enzyme recognizes a specific sequence, such as apeptide for Factor Xa or enterokinase. Examples of linkers include, butare not limited to, disulfide linkers, acid labile linkers (includingdialkoxybenzyl linkers), Sieber linkers, indole linkers, t-butyl Sieberlinkers, electrophilically cleavable linkers, nucleophilically cleavablelinkers, photocleavable linkers, cleavage under reductive conditions,oxidative conditions, cleavage via use of safety-catch linkers, andcleavage by elimination mechanisms.

Electrophilically cleaved linkers are typically cleaved by protons andinclude cleavages sensitive to acids. Suitable linkers include themodified benzylic systems such as trityl, p-alkoxybenzyl esters andp-alkoxybenzyl amides. Other suitable linkers includetert-butyloxycarbonyl (Boc) groups and the acetal system. The use ofthiophilic metals, such as nickel, silver or mercury, in the cleavage ofthioacetal or other sulphur-containing protecting groups can also beconsidered for the preparation of suitable linker molecules.

For nucleophilic cleavage, groups such as esters that are labile inwater (i.e., can be cleaved simply at basic pH) and groups that arelabile to non-aqueous nucleophiles, can be used. Fluoride ions can beused to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or t-butyldimethyl silane (TBDMS).

A linker susceptible to reductive cleavage may be used such as withdisulphide bond reduction. Catalytic hydrogenation using palladium-basedcatalysts has been used to cleave benzyl and benzyloxycarbonyl groups.

Oxidation-based approaches are well known in the art. These includeoxidation of p-alkoxybenzyl groups and the oxidation of sulphur andselenium linkers. Aqueous iodine to cleave disulphides and other sulphuror selenium-based linkers may also be used.

Safety-catch linkers are those that cleave in two steps. In a preferredsystem the first step is the generation of a reactive nucleophiliccenter followed by a second step involving an intra-molecularcyclization that results in cleavage. For example, levulinic esterlinkages can be treated with hydrazine or photochemistry to release anactive amine, which can then be cyclised to cleave an ester elsewhere inthe molecule (Burgess et al., J. Org. Chem. 62:5165-5168, 1997).

Elimination reactions may also be used. For example, the base-catalysedelimination of groups such as Fmoc and cyanoethyl, andpalladium-catalysed reductive elimination of allylic systems, may beused.

Where the linker is a thermal cleavable linker or thermal-sensitivelinker, the cleavage agent may be localized temperature elevation abovea threshold to disrupt or cleave the thermal cleavable group. In someembodiments, the temperature may be elevated in a localized region usingmicrowave irradiation to induce particle hyperthermia. Particlehyperthermia methods may be used, such as those reviewed in Dutz andHergt (Nanotechnology, 25:452001 (2014)) and described in U.S. Pat. No.7,718,445, U.S. Patent Publication No. 20030082633, International PatentPublication No. WO 2002029076, and U.S. Patent Publication No.20020197645, which are each incorporated herein by reference. In someembodiments, the temperature elevation may be achieved photothermally bytransferring energy from light to an absorbing target, such as a dye,pigment, or water. In one aspect, the source of light is a laser. Insome embodiments, the elevated temperature may cause the thermalseparation of double-stranded DNA.

g) Nanopore layer

In the present disclosure, detecting and/or counting the tag (e.g.,polymer, aptamer, nanoparticle) may be carried out by translocating thetag through or across a nanopore or nanochannel. In some embodiments,detecting and/or counting the tag (e.g., polymer, aptamer, nanoparticle)may be carried out by translocating the tag through or across at leastone or more nanopores or nanochannels. In some embodiments, at least toor more nanopores or nanochannels are presented side by side or inseries. In some embodiments, the nanopore or nanochannel is dimensionedfor translocation of not more than one tag at a time. Thus, thedimensions of the nanopore in some embodiments will typically depend onthe dimensions of the tag to be examined. A tag with a double-strandedregion can require a nanopore dimension greater than those sufficientfor translocation of a tag which is entirely single-stranded. Inaddition, a nanoparticle tag such as a nanobead tag can require largerpores or channels than oligomer tags. Typically, a pore of about 1 nmdiameter can permit passage of a single stranded polymer, while poredimensions of 2 nm diameter or larger will permit passage of adouble-stranded nucleic acid molecule. In some embodiments, the nanoporeor nanochannel is selective for a single stranded tag (e.g., from about1 nm to less than 2 nm diameter) while in other embodiments, thenanopore or nanochannel is of a sufficient diameter to permit passage ofdouble stranded polynucleotides (e.g., 2 nm or larger). The chosen poresize provides an optimal signal-to noise ratio for the analyte ofinterest.

In some embodiments, the pore may be between about 0.1 nm and about 1000nm in diameter, between about 50 nm and about 1000 nm, between about 100nm and 1000 nm, between about 0.1 nm and about 700 nm, between about 50nm and about 700 nm, between about 100 nm and 700 nm, between about 0.1nm and about 500 nm, between about 50 nm and about 500 nm, or betweenabout 100 nm and 500 nm. For example, the pore may be about 0.1 nm,about 0.2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm,about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1.0 nm, about 1.5 nm,about 2.0 nm, about 2.5 nm, about 3.0 nm, about 3.5 nm, about 4.0 nm,about 4.5 nm, about 5.0 nm, about 7.5 nm, about 10 nm, about 15 nm,about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm,about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about3500 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about850 nm, about 900 nm, about 950 nm, or about 1000 nm in diameter.

In general, nanopores are shorter in length than nanochannels. Ananochannel is substantially longer than a nanopore and may be useful inapplications where increasing the time it takes for a molecule totranslocate through it (as compared to the time for translocatingthrough or across a nanopore of the same diameter) is desirable. Lengthof a nanopore may range from about 0.1 nm to less than about 200 nm.Length of a nanochannel may range from about 500 nm to about 100 μm, orlonger. The diameter of a nanopore and a nanochannel may be similar.

Various types of nanopores may be used for analyzing the tags/aptamer.These include, among others, biological nanopores that employ abiological pore or channel embedded in a membrane. Another type ofnanopore layer is a solid state nanopore in which the channel or pore ismade whole or in part from a fabricated or sculpted solid statecomponent, such as silicon. In some embodiments, the nanopore is a solidstate nanopore produced using controlled dielectric breakdown. In someembodiments, the nanopore is a solid state nanopore produced by a methodother than controlled dielectric breakdown.

In certain embodiments, the length of a nanopore may be up to about 200nm, e.g., from about 0.1 nm to about 30 nm, from about 10 to about 80nm, from about 1 to about 50 nm, from about 0.1 nm to about 0.5 nm, fromabout 0.3 nm to about 1 nm, from about 1 nm to about 2 nm, from about0.3 nm to about 10 nm, or from about 10 to about 30 nm. The number ofnanopores in a nanopore layer may be about 1, 2, 3, 4, 5, 10, 30, 100,300, 1000, 3000, 10000, 30000, 100000, 300000 or more. The distancebetween nanopores in a layer between center to center may be about 100nm to about 300 nm, about 300 nm to about 500 nm, about 500 nm to about1000 nm, for example, 100 nm, 150 nm, 200 nm, or 300 nm.

In certain embodiments, multiple nanopore layers, each containing on ormore nanopores, can be arranged in series with each other, for detectingand/or counting the tag (e.g., polymer, aptamer, nanoparticle). In thiscase, detecting and/or counting the tag may be carried out bytranslocating the tag through or across each nanopore layer. As such,counting the number of tags translocating through or across a nanoporein a layer/sheet/membrane refers to counting multiple tags translocatingthrough or across one or more nanopores in one or morelayer/sheet/membrane. In certain embodiments, when more than onenanopore layers are present (e.g., one, two, three, four, five, six, orother number of nanopore layers as technically feasible), optionallythey are present in series wherein at least one nanopore in one layer isseparate from or stacked onto (e.g., above or on top of) anothernanopore in another layer, etc.). Where the nanopore layers are inseries, at least two electrodes can be used to create an electric fieldto drive tags through the pores and, optionally, additional electrodespositioned between the nanopore layers can further provide drivingcurrent.

i) Biological Pores

For detecting and, optionally, counting the tags/aptamer, any biologicalpore with channel dimensions that permit translocation of the tags canbe used. Two broad categories of biological channels are suitable forthe methods disclosed herein. Non-voltage gated channels allow passageof molecules through the pore without requiring a change in the membranepotential to activate or open the channel. On the other hand, voltagegated channels require a particular range of membrane potential toactivate channel opening. Most studies with biological nanopores haveused a-hemolysin, a mushroom-shaped homo-oligomeric heptameric channelof about 10 nm in length found in Staphylococcus aureus. Each subunitcontributes two beta strands to form a 14 strand anti-parallel betabarrel. The pore formed by the beta barrel structure has an entrancewith a diameter of approximately 2.6 nm that contains a ring of lysineresidues and opens into an internal cavity with a diameter of about 3.6nm. The stem of the hemolysin pore, which penetrates the lipid bilayer,has an average inside diameter of about 2.0 nm with a 1.5 nmconstriction between the vestibule and the stem. The dimensions of thestem are sufficient for passage of single-stranded nucleic acids but notdouble-stranded nucleic acids. Thus, a-hemolysin pores may be used as ananopore selective for single-stranded polynucleotides and otherpolymers of similar dimensions.

In other embodiments, the biological nanopore is of a sufficientdimension for passage of polymers larger than a single-stranded nucleicacid. An exemplary pore is mitochondrial porin protein, a voltagedependent anion channel (VDAC) localized in the mitochondrial outermembrane. Porin protein is available in purified form and, whenreconstituted into artificial lipid bilayers, generates functionalchannels capable of permitting passage of double-stranded nucleic acids(Szabo et al., 1998, FASEB J. 12:495-502). Structural studies suggestthat porin also has a beta-barrel type structure with 13 or 16 strands(Rauch et al., 1994, Biochem Biophys Res Comm 200:908-915). Porindisplays a larger conductance compared conductance of pores formed byα-hemolysin, maltoporin (LamB), and gramicidin. The larger conductanceproperties of porin support studies showing that the porin channel issufficiently dimensioned for passage of double-stranded nucleic acids.Pore diameter of the porin molecule is estimated at 4 nm. The diameterof an uncoiled double-stranded nucleic acid is estimated to be about 2nm.

Another biological channel that may be suitable for scanning doublestranded polynucleotides are channels found in B. subtilis (Szabo etal., 1997, J. Biol. Chem. 272:25275-25282). Plasma membrane vesiclesmade from B. subtilis and incorporated into artificial membranes allowpassage of double-stranded DNA across the membrane. Conductance of thechannels formed by B. subtilis membrane preparations is similar to thoseof mitochondrial porin. Although there is incomplete characterization(e.g., purified form) of these channels, it is not necessary to havepurified forms for the purposes herein. Diluting plasma membranepreparations, either by solubilizing in appropriate detergents orincorporating into artificial lipid membranes of sufficient surfacearea, can isolate single channels in a detection apparatus. Limiting theduration of contact of the membrane preparations (or proteinpreparations) with the artificial membranes by appropriately timedwashing provides another method for incorporating single channels intothe artificial lipid bilayers. Conductance properties may be used tocharacterize the channels incorporated into the bilayer.

In certain cases, the nanopores may be hybrid nanopores, where abiological pore is introduced in a solid state nanopore, e.g., ananopore fabricated in a non-biological material. For example,α-haemolysin pore may be inserted into a solid state nanopore. Incertain cases, the nanopores may be a hybrid nanopore described in Hallet al., Nature Nanotechnology, 28 Nov. 2010, vol. 5, pg. 874-877.

ii) Solid State Pores

In other embodiments, analysis of the tags is carried out bytranslocating the tag through or across a nanopore or nanochannelfabricated from non-biological materials. Nanopores or nanochannels canbe made from a variety of solid state materials using a number ofdifferent techniques, including, among others, chemical deposition,electrochemical deposition, electroplating, electron beam sculpting, ionbeam sculpting, nanolithography, chemical etching, laser ablation,focused ion beam, atomic layer deposition, and other methods well knownin the art (see, e.g., Li et al., 2001, Nature 412:166-169; and WO2004/085609).

In particular embodiments, the nanopores may be the nanopores describedin WO13167952A1 or WO13167955A1. As described in WO13167952A1 orWO13167955A1, nanopores having an accurate and uniform pore size may beformed by precisely enlarging a nanopore formed in a membrane. Themethod may involve enlarging a nanopore by applying a high electricpotential across the nanopore; measuring current flowing through thenanopore; determining size of the nanopore based in part on the measuredcurrent; and removing the electric potential applied to the nanoporewhen the size of the nanopore corresponds to a desired size. In certaincases, the applied electric potential may have a pulsed waveformoscillating between a high value and a low value, the current flowingthrough the nanopore may be measured while the electric potential isbeing applied to the nanopore at a low value.

Solid state materials include, by way of example and not limitation, anyknown semiconductor materials, insulating materials, and metals coatedwith insulating material. Thus, at least part of the nanopore(s) maycomprise without limitation silicon, silica, silicene, silicon oxide,graphene, silicon nitride, germanium, gallium arsenide, or metals, metaloxides, and metal colloids coated with insulating material.

To make a pore of nanometer dimensions, various feedback procedures canbe employed in the fabrication process. In embodiments where ions passthrough a hole, detecting ion flow through the solid state materialprovides a way of measuring pore size generated during fabrication (see,e.g., U.S. Published Application No. 2005/0126905). In otherembodiments, where the electrodes define the size of the pore, electrontunneling current between the electrodes gives information on the gapbetween the electrodes. Increases in tunneling current indicate adecrease in the gap space between the electrodes. Other feedbacktechniques will be apparent to the skilled artisan.

In some embodiments, the nanopore is fabricated using ion beamsculpting, as described in Li et al., 2003, Nature Materials 2:611-615.In some embodiments, the nanopore is fabricated using high current, asdescribed in WO13167952A1 or WO13167955A1. In other embodiments, thenanopores may be made by a combination of electron beam lithography andhigh energy electron beam sculpting (see, e.g., Storm et al., 2003,Nature Materials 2:537-540). A similar approach for generating asuitable nanopore by ion beam sputtering technique is described in Henget al., 2004, Biophy J 87:2905-2911. The nanopores are formed usinglithography with a focused high energy electron beam on metal oxidesemiconductor (CMOS) combined with general techniques for producingultrathin films. In other embodiments, the nanopore is constructed asprovided in U.S. Pat. Nos. 6,627,067; 6,464,842; 6,783,643; and U.S.Publication No. 2005/0006224 by sculpting of silicon nitride.

In some embodiments, the nanochannels can be constructed as a gold orsilver nanotube. These nanochannels are formed using a template ofporous material, such as polycarbonate filters prepared using a tracketch method, and depositing gold or other suitable metal on the surfaceof the porous material. Track etched polycarbonate membranes aretypically formed by exposing a solid membrane material to high energynuclear particles, which creates tracks in the membrane material.Chemical etching is then employed to convert the etched tracks to pores.The formed pores have a diameter of about 10 nm and larger. Adjustingthe intensity of the nuclear particles controls the density of poresformed in the membrane. Nanotubes are formed on the etched membrane bydepositing a metal, typically gold or silver, into the track etchedpores via an electroless plating method (Menon et al., 1995, Anal Chem67:1920-1928). This metal deposition method uses a catalyst deposited onthe surface of the pore material, which is then immersed into a solutioncontaining Au(I) and a reducing agent. The reduction of Au(I) tometallic Au occurs on surfaces containing the catalyst. Amount of golddeposited is dependent on the incubation time such that increasing theincubation time decreases the inside diameter of the pores in the filtermaterial. Thus, the pore size may be controlled by adjusting the amountof metal deposited on the pore. The resulting pore dimension is measuredusing various techniques, for instance, gas transport properties usingsimple diffusion or by measuring ion flow through the pores using patchclamp type systems. The support material is either left intact, orremoved to leave gold nanotubes. Electroless plating technique iscapable of forming pore sizes from less than about 1 nm to about 5 nm indiameter, or larger as required. Gold nanotubes having pore diameter ofabout 0.6 nm appears to distinguish between Ru(bpy)2+2 and methylviologen, demonstrating selectivity of the gold nanopores (Jirage etal., 1997, Science 278:655-658). Modification of a gold nanotube surfaceis readily accomplished by attaching thiol containing compounds to thegold surface or by derivatizing the gold surface with other functionalgroups. This features permits attachment of pore modifying compounds aswell as sensing labels, as discussed herein. Devices, such as thecis/trans apparatuses used for biological pores described herein, can beused with the gold nanopores to analyze single coded molecules.

Where the mode of detecting the tag involves current flow through thetag (e.g., electron tunneling current), the solid state membrane may bemetalized by various techniques. The conductive layer may be depositedon both sides of the membrane to generate electrodes suitable forinterrogating the tag along the length of the chain, for example,longitudinal electron tunneling current. In other embodiments, theconductive layer may be deposited on one surface of the membrane to formelectrodes suitable for interrogating tag across the pore, for example,transverse tunneling current. Various methods for depositing conductivematerials are known, including, sputter deposition (i.e., physical vapordeposition), non-electrolytic deposition (e.g., colloidal suspensions),and electrolytic deposition. Other metal deposition techniques arefilament evaporation, metal layer evaporation, electron-beamevaporation, flash evaporation, and induction evaporation, and will beapparent to the skilled artisan.

In some embodiments, the detection electrodes are formed by sputterdeposition, where an ion beam bombards a block of metal and vaporizesmetal atoms, which are then deposited on a wafer material in the form ofa thin film. Depending on the lithography method used, the metal filmsare then etched by means of reactive ion etching or polished usingchemical-mechanical polishing. Metal films may be deposited on preformednanopores or deposited prior to fabrication of the pore.

In some embodiments, the detection electrodes are fabricated byelectrodeposition (see, e.g., Xiang et al., 2005, Angew. Chem. Int. Ed.44:1265-1268; Li et al., Applied Physics Lett. 77(24):3995-3997; andU.S. Publication Application No. 2003/0141189). This fabrication processis suitable for generating a nanopore and corresponding detectionelectrodes positioned on one face of the solid state film, such as fordetecting transverse electron tunneling. Initially, a conventionallithographic process is used to form a pair of facing electrodes on asilicon dioxide layer, which is supported on a silicon wafer. Anelectrolyte solution covers the electrodes, and metal ions are depositedon one of the electrodes by passing current through the electrode pair.Deposition of metal on the electrodes over time decreases the gapdistance between the electrodes, creating not only detection electrodesbut a nanometer dimensioned gap for translocation of coded molecules.The gap distance between the electrodes may be controlled by a number offeedback processes.

Where the detection is based on imaging of charge induced field effects,a semiconductor can be fabricated as described in U.S. Pat. No.6,413,792 and U.S. published application No. 2003/0211502. The methodsof fabricating these nanopore devices can use techniques similar tothose employed to fabricate other solid state nanopores.

Detection of the tag, such as a polynucleotide, is carried out asfurther described below. For analysis of the tag, the nanopore may beconfigured in various formats. In some embodiments, the device comprisesa membrane, either biological or solid state, containing the nanoporeheld between two reservoirs, also referred to as cis and trans chambers(see, e.g., U.S. Pat. No. 6,627,067). A conduit for electron migrationbetween the two chambers allows electrical contact of the two chambers,and a voltage bias between the two chambers drives translocation of thetag through the nanopores. A variation of this configuration is used inanalysis of current flow through nanopores, as described in U.S. Pat.Nos. 6,015,714 and 6,428,959; and Kasianowiscz et al., 1996, Proc NatlAcad Sci USA 93:13770-13773, the disclosures of which are incorporatedherein by reference.

Variations of above the device are disclosed in U.S. applicationpublication no. 2003/0141189. A pair of nanoelectrodes, fabricated byelectrodeposition, is positioned on a substrate surface. The electrodesface each other and have a gap distance sufficient for passage of asingle nucleic acid. An insulating material protects the nanoelectrodes,exposing only the tips of the nanoelectrodes for the detection of thenucleic acid. The insulating material and nanoelectrodes separate achamber serving as a sample reservoir and a chamber to which the polymeris delivered by translocation. Cathode and anode electrodes provide anelectrophoresis electric field for driving the tag from the samplechamber to the delivery chamber.

The current bias used to drive the tag through the nanopore can begenerated by applying an electric field directed through the nanopore.In some embodiments, the electric field is a constant voltage orconstant current bias. In other embodiments, the movement of the tag iscontrolled through a pulsed operation of the electrophoresis electricfield parameters (see, e.g., U.S. Patent Application No. 2003/141189 andU.S. Pat. No. 6,627,067). Pulses of current may provide a method ofprecisely translocating one or only a few bases of an oligonucleotidetag for a defined time period through the pore and to briefly hold thetag within the pore, and thereby provide greater resolution of theelectrical properties of the tag.

The nanopore devices may further comprise an electric or electromagneticfield for restricting the orientation of the oligonucleotide tag as itpasses through the nanopore. This holding field can be used to decreasethe movement of the oligonucleotide tag within the pore. In someembodiments, an electric field that is orthogonal to the direction oftranslocation is provided to restrict the movement of the tag moleculewithin the nanopore. This is illustrated in U.S. Application PublicationNo. 2003/0141189 through the use of two parallel conductive plates aboveand beneath the sample plate. These electrodes generate an electricfield orthogonal to the direction of translocation of a tag molecule,and thus holding the tag molecule to one of the sample plates. Anegatively charged backbone of a DNA, or nucleic acid modified to havenegative charges on one strand, will be oriented onto the anodic plate,thereby limiting the motion of the tag molecule.

In still other embodiments, controlling the position of the tag iscarried out by the method described in U.S. Application Publication No.2004/0149580, which employs an electromagnetic field created in the porevia a series of electrodes positions near or on the nanopore. In theseembodiments, one set of electrodes applies a direct current voltage andradio frequency potential while a second set of electrodes applies anopposite direct current voltage and a radio frequency potential that isphase shifted by 180 degrees with respect to the radio frequencypotential generated by the first set of electrodes. This radio frequencyquadrupole holds a charged particle (e.g., nucleic acid) in the centerof the field (i.e., center of the pore).

In exemplary embodiments, the nanopore membrane may be a multilayerstack of conducting layers and dielectric layers, where an embeddedconducting layer or conducting layer gates provides well-controlled andmeasurable electric field in and around the nanopore through which thetag translocates. In an aspect, the conducting layer may be graphene.Examples of stacked nanopore membranes are found in US20080187915 andUS20140174927, for example.

It is understood that the nanopore may be located in a membrane, layeror other substrate, which terms have been used interchangeably todescribe a two-dimensional substrate comprising a nanopore.

In certain embodiments, the nanopore may be formed as part of the assayprocess for detecting and/or determining concentration of an analyteusing the nanopore. Specifically, a device for detecting and/ordetermining concentration of an analyte using a nanopore may initiallybe provided without a nanopore formed in a membrane or layer. The devicemay include a membrane separating two chambers on the opposite sides ofthe membrane (a cis and a trans chamber). The cis and the trans chambersmay include a salt solution and may be connected to a source ofelectricity. When a nanopore is to be created in the membrane, a voltageis applied to the salt solution in the cis and trans chamber andconductance through the membrane measured. Prior to the creation of ananopore, there is no or minimal current measured across the membrane.Following creation of a nanopore, the current measured across themembrane increases. The voltage may be applied for an amount of timesufficient to create a nanopore of the desired diameter. Following thecreation of a nanopore, an analyte or tag may be translocated throughthe nanopore and the translocation event detected. In certainembodiments, the same salt solution may be used for nanopore creation aswell as for detection of translocation of an analyte or tag through thenanopore. Any suitable salt solution may be utilized for nanoporecreation and/or translocation of an analyte or tag through the nanopore.Any salt solution that does not damage the counting label can be used.Exemplary salt solutions include lithium chloride, potassium chloride,sodium chloride, calcium chloride, magnesium chloride and the like. Theconcentration of the salt solution may be selected based on the desiredconductivity of the salt solution. In certain embodiments, the saltsolution may have a concentration ranging from 1 mM to 10 M, e.g., 10mM-10 M, 30 mM-10 M, 100 mM-10 M, 1 M-10 M, 10 mM-5 M, 10 mM-3 M, 10mM-1 M, 30 mM-5 M, 30 mM-3 M, 30 mM-1 M, 100 mM-5 M, 100 mM-3 M, 100mM-1 M, 500 mM-5 M, 500 mM-3 M, or 500 mM-1 M, such as, 10 mM, 30 mM,100 mM, 500 mM, 1 M, 3 M, 5 M, or 10 M.

In some embodiments, the nanopore may become blocked, and the blockednanopore is cleared by modulating the pattern of voltage applied by theelectrodes across the nanopore layer or membrane. In some cases, ablocked nanopore is cleared by reversing polarity of the voltage acrossthe nanopore layer or membrane. In some cases, a blocked nanopore iscleared by increasing the magnitude of the voltage applied across thenanopore layer or membrane. The increase in voltage may be transitoryincrease, lasting 10 seconds (s) or less, e.g., 8 s or less, 6 s orless, 5 s or less, 4 s or less, 3 s or less, 2 s or less, 1 s or less,0.5 s or less, 0.4 s or less, 0.3 s or less, 0.2 s or less, including0.1 s or less.

h) Signal Detection

Interrogating the tag/aptamer by translocation through or across ananopore and detecting the detectable property generates a signal thatcan be used to count (i.e., determine the quantity or concentration)and/or identify (i.e., determine the presence of) the tag/aptamer. Thetype of detection method employed may correspond to the property beingdetected for the tags.

In some embodiments, the detectable property is the effect of the tag onthe electrical properties of the nanopore as the tag translocatesthrough the pore. Electrical properties of the nanopore include amongothers, current amplitude, impedance, duration, and frequency. Incertain cases, the tag may be identified by using nanopore forcespectroscopy (see e.g., Tropini C. and Marziali A., Biophysical Journal,2007, Vol. 92, 1632-1637). Devices for detecting the pore's electricalproperties may include a nanopore incorporated into a layer such as, athin film or a membrane, where the film or membrane separates a cischamber and a trans chamber connected by a conducting bridge. The tag tobe analyzed may be present on the cis side of the nanopore in an aqueoussolution typically comprising one or more dissolved salts, such aspotassium chloride. Application of an electric field across the poreusing electrodes positioned in the cis and trans side of the nanoporecauses translocation of the tag through the nanopore, which affects themigration of ions through the pore, thereby altering the pore'selectrical properties. Current may be measured at a suitable timefrequency to obtain sufficient data points to detect a current signalpattern. The generated signal pattern can then be compared to a set ofreference patterns in which each reference pattern is obtained fromexamination of a single population of known tags bound to analyte in asample with a known analyte concentration. As previously noted, thenumber of tags of the same type translocating though a nanopore(s) maybe counted per unit time, such as, the number of tags of the same typetranslocating through or across nanopore(s) per 15 min, 13 min, 10 min,8 min, 6 min, 4 min, 2 min, 1 min, 30 sec, per 20 sec, per 15 sec, per10 sec, per 5 sec, per 1 sec, per 100 millisec, per 10 millisec, or per1 millisec. In some cases, the number of tags of the same typetranslocating though a nanopore(s) may be counted for a certain periodof time to determine the amount of time to reach a threshold count.Shifts in current amplitude, current duration, current frequency, andcurrent magnitude may define a signal pattern for the tag and may beused to distinguish different tags from each other. Measurement ofcurrent properties of a nanopore, such as by patch clamp techniques, isdescribed in publications discussed above and in various referenceworks, for example, Hille, B, 2001, Ion Channels of Excitable Membranes,3rd Ed., Sinauer Associates, Inc., Sunderland, Mass. The number ofcounts measured over a time period (counts/time) is proportional to theconcentration of the molecule (e.g., tag) translocating through oracross the nanopore. The concentration of the tag may be determined bygenerating a standard curve. For example, a series of differentconcentrations of a standard molecule may be translocated through ananopore and the counts/time measured to calculate a count rate for eachconcentration. The count rate of the tag being measured would becompared to the standard curve to calculate the concentration of thetag.

In some embodiments, the detectable property of the tag may be quantumtunneling of electrons. Quantum tunneling is the quantum-mechanicaleffect of transitioning through a classically-forbidden energy state viaa particle's quantum wave properties. Electron tunneling occurs where apotential barrier exists for movement of electrons between a donor andan acceptor. To detect electron tunneling, a microfabricated electrodetip may be positioned about 2 nanometers from the specimen. At anappropriate separation distance, electrons tunnel through the regionbetween the tip and the sample, and if a voltage is applied between thetip and the sample, a net current of electrons (i.e., tunneling current)flows through the gap in the direction of the voltage bias. Where thenanodevice uses detection electrodes for measuring tunneling current,the electrodes are positioned proximately to the translocating tag suchthat there is electron tunneling between the detection electrodes andtag. As further discussed below, the arrangement of the electrodesrelative to the translocating tag may dictate the type of electrontransport occurring through the tag.

In some embodiments, analysis of the tag may involve detecting currentflow occurring through the nucleic acid chain (i.e., longitudinallyalong the nucleic acid chain) (Murphy et al., 1994, Proc Natl Acad SciUSA 91(12):5315-9). The exact mechanism of electron transfer is unknown,although electron tunneling is given as one explanation for DNA'stransport properties. However, the physics underlying electron transportthrough a double-stranded nucleic acid is not limiting for the purposesherein, and detection of current flowing through the nucleic acid servesto distinguish one polymer tag from another polymer tag. For detectionof electron flow occurring longitudinally through the tag moleculechain, the detection electrodes may be positioned longitudinally to thedirection of tag molecule translocation such that there is a gap betweenthe electrodes parallel to the chain of an extended tag molecule. Invarious embodiments, the detection electrodes may be placed on oppositesides of a layer(s) (e.g., membrane) separating the two sides of thenanopore, while in other embodiments, the detection electrodes may bepositioned within the layer(s) that separate the two sides of thenanopore.

Another mode of electron flow in a nucleic acid is that occurring acrossthe nucleic acid, for example, a direction transverse to an extendednucleic acid chain (e.g., across the diameter of a double-strandednucleic acid). In a double-stranded nucleic acid, electron transport mayoccur through the paired bases while in a single-stranded nucleic acid,electron transport may occur through a single unpaired base.Furthermore, differences in the chemical compositions, hydrationstructures, interactions with charged ions, spatial orientation of eachbase, and different base pairing combinations may alter the transverseelectron transport characteristics, and thus provide a basis fordistinguishing tag molecules that differ in sequence and/or polymerbackbone. For detection of electron flow across a tag molecule (i.e.,transverse to an extended nucleic acid chain), the detection electrodesare positioned on one side of the nanopore to interrogate the tagmolecule across rather than through the nanopore.

In embodiments of longitudinal or transverse detection, the thickness ofthe electrodes may determine the total number of bases interrogated bythe electrodes. For transverse detection, the tips of the detectionelectrodes may be dimensioned to interrogate a single nucleobase (asdefined herein), and thereby obtain single base resolution. In otherembodiments, the dimensions of the detection electrode are arranged tointerrogate more than one nucleobase. Thus, in some embodiments, thenumber of nucleobases interrogated at any one time may be about 2 ormore, about 5 or more, about 10 or more, or about 20 or more dependingon the resolution required to detect differences in the various polymersequences of the tag molecule.

In other embodiments, differences in the structure of a tag may bedetected as differences in capacitance. This type of measurement isillustrated in US2003/0141189. Capacitance causes a phase shift in anapplied ac voltage at a defined applied frequency and impedance. Phaseshift characteristics for each nucleobase is determined for nucleicacids of known sequence and structure, and used as reference standardsfor identifying individual base characteristics. Nearest neighboranalysis may permit capacitance measurements extending to more than asingle nucleobase.

In other embodiments, the detection technique may be based on imagingcharge-induced fields, as described in U.S. Pat. No. 6,413,792 and U.S.published application No. 2003/0211502, the disclosures of which areincorporated herein by reference. For detecting a tag based on chargeinduced fields, a semiconductor device described above is used.Application of a voltage between a source region and a drain regionresults in flow of current from the source to the drain if a channel forcurrent flow forms in the semi-conductor. Because each nucleobase has anassociated charge, passage of a tag molecule through the semiconductorpore induces a change in the conductivity of the semiconductor materiallining the pore, thereby inducing a current of a specified magnitude andwaveform. Currents of differing magnitude and waveform are produced bydifferent bases because of differences in charge, charge distribution,and size of the bases. In the embodiments disclosed in U.S. Pat. No.6,413,792, the polymer passes through a pore formed of a p-type siliconlayer. Translocation of the tag molecule is achieved by methods similarto those used to move a polymer through other types of channels, asdescribed above. The magnitude of the current is expected to be on theorder of microampere range, which is much higher than the expectedpicoampere currents detected by electron tunneling. Because the polymerblock regions in the tag molecule comprise more than a singlenucleobase, these block polymer regions should produce distinctivesignals reflective of the charge and charge distribution of the blockpolymer regions.

It is to be understood that although descriptions above relate toindividual detection techniques, in some embodiments, a plurality ofdifferent techniques may be used to examine a single tag molecule (see,e.g., Kassies et al., 2005, J Microsc 217:109-16). Examples of multipledetection modes include, among others, current blockade in combinationwith electron tunneling current, and current blockage in combinationwith imaging charge induced fields. Concurrent detection with differentdetection modes may be used to identity a tag molecule by correlatingthe detection time of the resulting signal between different detectionmodes.

In some embodiments, measuring the number of tags translocating throughthe layer or detecting tags translocating through the layer includesobserving a current blockade effect of the tags on the nanopores. Insome embodiments, an analyte is present in the sample when the currentblockade effect is above a threshold level.

3. DEVICE FOR ANALYTE ANALYSIS

The present disclosure describes a microfluidics device used inconjunction with a nanopore device and an integrated microfluidicsnanopore device. The disclosed microfluidics device used in conjunctionwith a nanopore device and an integrated microfluidics nanopore devicemay be used in the method of analyte analysis, as described above.However, in certain cases, the devices described herein may be used forother applications. Likewise, in certain cases, the methods describedherein may be used with other devices.

A microfluidics device used in conjunction with a nanopore device isdepicted in FIGS. 1A and 1B. The microfluidics device 10 is depictedwith a fluid droplet 11 which is to be analyzed in the nanopore device15. The fluid droplet may include a tag (e.g., a cleaved tag or anaptamer) that is to be counted using the nanopore device. The nanoporedevice 15 includes a first chamber 16, a layer 17 with a nanopore 18,and a second chamber 19. FIGS. 1A and 1B depict a liquid transfer step 1in which the fluid droplet 11 is removed from the microfluidics device10 and placed into the nanopore device 15. As depicted in FIG. 1A, thefluid droplet 11 is deposited over the layer 17 in a manner that resultsin the droplet being split apart across the layer 17 and positioned atthe nanopore 18. The fluid droplet may be introduced into the nanoporedevice 15 via an entry port (not shown). The entry port may bepositioned over a section of the layer 17. For example, the entry portmay be located in an opening in a wall of a chamber in which the layercontaining nanopore is positioned. In FIG. 1B, the liquid droplet 11 isdeposited in the first chamber 16. A buffer addition step 2, introducesa buffer in the second chamber 19. In other embodiments, buffer may beadded to the second chamber 19 prior to the introduction of the liquiddroplet 11 into the first chamber 16. In yet other embodiments, theliquid droplet 11 may be deposited in the second chamber 19 before orafter buffer is added to the first chamber 16. In FIG. 1A, a step ofaddition of a buffer to either chamber is not needed.

In another embodiment, the device may be an integrated device. Theintegrated device may include a microfluidics module and a nanoporemodule that may be built separately and then combined to form theintegrated device or the microfluidics module and the nanopore modulemay be built-in together in a single device.

FIGS. 2A and 2B depict a schematic of an integrated device that has amicrofluidics module combined with a nanopore module and the two modulesare integrated by connecting them using a channel. Although FIGS. 2A and2B depict a device that includes individual modules that are combined togenerate an integrated device, it is understood that the device of FIGS.2A and 2B can also be manufactured as a unitary device in which the twomodules are connected.

In FIGS. 2A and 2B, top panel, a microfluidics module 20 is depictedwith a fluid droplet 25 which is to be analyzed in the nanopore device30. The nanopore module 30 includes a first chamber 31, a layer 32 witha nanopore 33, and a second chamber 34. The microfluidics module 20 isintegrated with the nanopore module 30 via a channel 40. The channelfluidically connects the two modules and facilitates the movement of thedroplet 25 from the microfluidics module 20 to the nanopore module 30.The middle panel illustrates the movement of the droplet 25 from themicrofluidics module 20 to the nanopore module 30 via the channel 40. Asshown in FIG. 2A, the channel may connect the microfluidics module 20 toan entry port in the nanopore module 30. The entry port (not shown) maybe positioned such that the fluid droplet 25 is deposited over the layer32 in a manner that results in the droplet being split apart across thelayer 32 and positioned at the nanopore 33. At the end of the transferprocess, the fluid droplet is positioned across the nanopore 33 (FIG.2A, bottom panel). In other embodiments, the channel 40 may connect themicrofluidics module 20 to an entry port in a first or second chamber ofthe nanopore module 30. Such an embodiment is shown in FIG. 2B, wherethe channel 40 connects the microfluidics module 20 to an entry port ina first chamber 31 of the nanopore module 30. Following or prior to thetransfer of the liquid droplet 25 into the first chamber 31, a buffermay be added to the second chamber. In step 2 of FIG. 2B, buffer isadded to the second chamber 34 following the transfer of the droplet 25to the first chamber 31. Optionally, after the transfer is completed,the channel 40 may be removed and the two modules separated. Themicrofluidics and nanopore devices and modules shown in FIGS. 1A, 1B, 2Aand 2B, respectively, are each individually functional.

FIGS. 2C-2H depicts an embodiment of an integrated device which includesa digital microfluidics module 50 and a nanopore module 60. The digitalmicrofluidics module is depicted with an array of electrodes 49 that areoperatively connected to a plurality of reagent reservoirs 51 used forgeneration of droplets to be transported to the nanopore module. One ormore of the reservoirs 51 may contain a reagent or a sample. Differentreagents may be present in different reservoirs. Also depicted in themicrofluidics module 50 are contact pads 53 that connect the array ofelectrodes 49 to a power source (not shown). Trace lines connecting thearray of electrodes 49 to the contact pads are not depicted. The arrayof electrodes 49 transport one or more droplets (such as buffer dropletor a droplet containing buffer and/or tag (e.g., cleaved tag ordissociated aptamer)) to one or both of the transfer electrodes 71 and72 located at the interface 100 between the digital microfluidics module50 and a nanopore module 60. The digital microfluidics module 50 and thenanopore module 60 are operatively connected at the interface 100. Thenanopore module 60 includes at least two microfluidic capillary channels61 and 62 that intersect with each other at the location at which ananopore layer 70 is disposed. The two microfluidic capillary channels61 and 62 are located in two different substrates in the nanopore module(depicted in FIG. 2D). Thus, the nanopore module includes a firstsubstrate 63 (e.g., bottom substrate) that includes a microfluidiccapillary channel 61 in a top surface of the first substrate 63 andfurther includes a second substrate 64 (e.g., top substrate) with amicrofluidic capillary channel 62 in the first surface of the secondsubstrate. The second substrate 64 overlays the microfluidic capillarychannel 61 and the first substrate 63 underlays the microfluidiccapillary channel 62. The capillary channel 62 overlays capillarychannel 61 at the point of intersection of the two channels at thelocation of the nanopore layer 70 (see also FIG. 2D, bottom panel). Thetwo capillary channels are physically separated at the intersection bythe nanopore layer 70 placed at the intersection. The nanopore layer 70includes at least one nanopore (not shown) that is positioned at theintersection of the capillary channels and allows transport of moleculesfrom one capillary channel to the other through the nanopore. Thecapillary channels 61 and 62 open at the interface 100 at a first endsof the capillary channels and open to a reservoir/vent (84 and 85, asseen in FIG. 2C) at the second ends of the capillary channels. Alsodepicted in FIG. 2C is a cover substrate 101 that is positioned over thearray of electrodes 49. The cover substrate 101 defines a gap in themicrofluidics module in which droplets are manipulated. The coversubstrate 101 may optionally include an electrode 55 (e.g., a referenceelectrode) disposed on a bottom surface of the cover substrate 101providing a bi-planar electrode configuration for manipulating dropletsin the microfluidics module 50. In absence of a bi-planar electrodeconfiguration, droplets may be manipulated in the microfluidics module50 by using coplanar electrode actuation, for example using the array ofelectrode 49 or another coplanar electrode configuration. For example,the coplanar electrodes described in U.S. Pat. No. 6,911,132 may be usedfor manipulating droplets in the microfluidics module 50.

FIG. 2D, top panel, shows a schematic of a front view of a cross-sectionof the interface 100 at which the digital microfluidics module 50 and ananopore module 60 are operatively connected. A schematic of a side viewof a cross-section of the device at the transfer electrode 72 isdepicted in the bottom panel of FIG. 2D. FIG. 2D, top panel shows twodroplets (65 a and 65 b) positioned on two transfer electrodes 71 and 72that are located at the interface 100 between microfluidics module 50and a nanopore module 60. As illustrated in FIG. 2D, top panel, thedroplet 65 a positioned at electrode 71 is aligned with the opening inthe capillary channel 61 while the droplet 65 b positioned at electrode72 is aligned with the opening in capillary channel 62. FIG. 2D, bottompanel illustrates a side view of a cross-section of the integrateddevice showing placement of droplet 65 b on transfer electrode 72. Thedroplet 65 b is positioned to move into the capillary channel 62Capillary channel 61 is also shown; however, the capillary channel is ata distance from the transfer electrode 72 and is aligned with transferelectrode 71 (not shown). The cover substrate 101 with an electrode 55disposed on the bottom surface of the cover substrate 101 is alsodepicted. In the embodiments of the integrated devices depicted in FIG.2D-2H, the nanopore module is disposed on the same substrate as theelectrode array of the microfluidics module.

The vertical distance between the top surface of the transfer electrodesand the entrance to the capillary channels may be determined by thethickness of the substrates forming the lower part of the microfluidicsmodule and the nanopore module. The vertical distance may be set basedon the volume of the droplets to be transferred to the nanopore module.The vertical distance may be adjusted by varying the thickness of thesubstrates. For example, the substrates (e.g., substrate 63) of thenanopore module may kept relatively thin or the thickness of thesubstrate on which the transfer electrodes are disposed can be increased(for example by using a thicker substrate) to ensure that the droplet isaligned with the entrance of the capillary channel. An exemplary devicein which the droplets are brought into alignment with the entrance tothe capillary channels by using a microfluidics module having a thickerbottom substrate is depicted in FIG. 2E. The device shown in FIG. 2E hasthe same configuration as described for FIGS. 2C-2D. However, thethickness of the substrate 59 a on which the electrode array ispositioned is increased relative to the thickness of the part of thesubstrate on which the nanopore module is disposed. FIG. 2E, top paneldepicts a front view of a cross section at the interface 100 between themicrofluidics module and the nanopore module. FIG. 2E, bottom paneldepicts a side view of a cross section at the position of the transferelectrode 72 and capillary channel 62. As illustrated in FIG. 2E, thesubstrate 59 a on which the electrode array 49 and the transferelectrodes 71 and 72 are disposed is thicker than the substrate 59 b onwhich the nanopore module is disposed. As shown in FIG. 2E, bottompanel, substrate 59 a has a first height H1 while substrate 59 b has asecond height H2, where H1 is greater than H2. The difference in heightbetween the substrates 59 a and 59 b results in alignment of thecapillary channels 61 and 62 in the nanopore module with the dropletspositioned on electrodes 71 and 72, respectively. Also depicted in thebottom panel of FIG. 2E is the channel 61. As evident from FIG. 2C,capillary channel 62 is perpendicular to the capillary channel 61 at thelocation of the nanopore layer 70. Channel 61 is aligned with thetransfer electrode 71 and is configured to receive droplet 65 apositioned on transfer electrode 71. While the two capillary channelsare depicted to be perpendicular to each other at the point ofintersection, other configurations are also envisioned where the twochannels intersect at an angle other than 90 degrees.

Upon contact with the capillary channel, the droplets move into thecapillary channel via any suitable means, such as, capillary action. Themovement of a droplet into the capillary channel may be facilitated byadditional methods/materials. For example, the droplets may move intothe capillary channel via diffusion, Brownian motion, convection,pumping, applied pressure, gravity-driven flow, density gradients,temperature gradients, chemical gradients, pressure gradients (positiveor negative), pneumatic pressure, gas-producing chemical reactions,centrifugal flow, capillary pressure, wicking, electric field-mediated,electrode-mediated, electrophoresis, dielectrophoresis, magnetophoresis,magnetic fields, magnetically driven flow, optical force, chemotaxis,phototaxis, surface tension gradient driven flow, Marangoni stresses,thermo-capillary convection, surface energy gradients, acoustophoresis,surface acoustic waves, electroosmotic flow, thermophoresis,electrowetting, opto-electrowetting, or combinations thereof. Inaddition or alternatively, movement of a droplet into the capillarychannel may be facilitated by using for example, an actuation force,such as those disclosed herein; using hydrophilic coating in thecapillary; varying size (e.g. width and/or height and/or diameter and/orlength) of the capillary channel).

In the embodiments depicted in FIGS. 2C-2H, the flow of a fluid acrosscapillaries channels 61 and 62 is controlled at least in part bychanging the cross-section of the capillaries—the fluid initially movesrelatively quickly till it enters a narrower portion of the capillaries.One or both droplets may be droplets containing analyte to be detectedor counted (or cleaved tag or dissociated aptamer) or conductivesolution (e.g., buffer not containing an analyte) for analysis via thenanopore. In certain cases, one droplet 65 a may be a droplet containingan analyte/tag/aptamer while the other droplet 65 b may be a bufferdroplet. While a single droplet is depicted for each channel, inpractice, multiple droplets may be transported to the nanopore module.For example, the multiple droplets may be transported to the nanoporemodule in a sequential manner. In some cases, multiple droplets may begathered at one or both transfer electrodes to generate a larger dropletwhich is transported to the nanopore module.

FIG. 2F illustrates an exemplary configuration of the various electrodesused in the integrated device. As noted above, a single continuouselectrode 55 (not shown in FIG. 2F) is positioned in a spaced apartmanner from the array of electrodes 49 in the microfluidics module 60.The array of electrodes includes a series of individually controllableelectrodes. The electrode 55 is disposed on a lower surface of the coversubstrate 101. Electrode 55 and the array of electrodes move thedroplets over to the transfer electrodes. While it is depicted thatelectrode 55 does not cover the transfer electrodes 71 and 72, incertain exemplary devices, the cover substrate 101 and the electrode 55may extend over the transfer electrodes. In embodiments where theelectrode 55 does not cover the transfer electrodes, co-planarelectrodes may be used to move droplets to the transfer electrode (e.g.,coplanar actuation as described in U.S. Pat. No. 6,911,132). Asdescribed herein, the single electrode 55 may serve as a reference or agrounding electrode, while the array of electrodes 49 may beindividually controllable (for example, the array of electrodes may beactuation electrodes that can be actuated independently). Electrodepairs: pair 80 a and 80 b and pair 90 a and 90 b are positioned in thenanopore module. Electrode pairs 80 a, 80 b and 90 a, 90 b are used toestablish opposite polarity across the nanopore layer 70 for drivingcharged molecules through the nanopore(s) in the nanopore layer 70. Insome embodiments, the electrode pair 80 a and 80 b may be positiveelectrodes and the electrode pair 90 a and 90 b may be negativeelectrodes. FIG. 2G illustrates an alternative electrode configurationfor the nanopore module where two electrodes 80 and 90 (instead of four)are used for establishing a polarity difference across the nanoporelayer 70. These examples demonstrate the use of either symmetrical (fourelectrodes) or asymmetrical (two electrodes) electrode configurationsthat generate an electric potential gradient across the nanopore layerfor translocating charged molecules through the nanopore.

FIG. 2H illustrates an alternative configuration of the capillarychannels where only one channel 61 is connected to the microfluidicsmodule at the interface 100. The other channel 62 is connected to tworeservoirs that may be filled with a conductive liquid to facilitatetransfer of charged molecules across the nanopore.

In certain cases, the integrated devices provided herein may befabricated by forming reservoirs and array of electrodes for the digitalmicrofluidics module portion on a first area of a top surface of a firstsubstrate. A second substrate may be prepared by disposing a singleelectrode (e.g., electrode 55) on the bottom surface of the secondsubstrate and positioned over the array of individually controllableelectrodes in a spaced apart manner to provide facing orientationbetween the single electrode and the array of electrodes for bi-planardroplet actuation. As used herein, “droplet actuation” refers tomanipulation of droplets using a microfluidics device as disclosedherein or using a droplet actuator as disclosed in U.S. Pat. Nos.6,911,132, 6,773,566, or U.S. Pat. No. 6,565,727, the disclosures ofwhich are incorporated herein by reference. Thus, the configuration ofthe bi-planar electrodes or the array of electrodes of the devicesdisclosed herein may be similar to those disclosed in U.S. Pat. Nos.6,911,132, 6,773,566, or U.S. Pat. No. 6,565,727. The electrode 55 onthe second substrate may also be referred to as a reference electrode.The electrodes in the microfluidics module may optionally be coated witha dielectric material. A hydrophobic coating may also be provided on thedielectric.

In certain embodiments, a microchannel may be formed on a thirdsubstrate which may be disposed on a second area of the first substrateon which the array of electrodes 49 is disposed. For example, a thirdsubstrate may be bonded onto a second area on the first substrate inwhich the microfluidics electrode array is disposed in the first area.The substrate may have a pre-formed microchannel or a microchannel maybe formed after the bonding step. A fourth substrate with a secondmicrochannel may be disposed on top of the substrate containing themicrochannel to provide an integrated device as depicted in FIG. 2C-2H.The nanopore layer may be disposed on either microchannel at thelocation of the intersection of the two microchannels. Thus, thesubstrates forming the nanopore module may include microchannels thatare open at either ends and on one side. The placement of the fourthsubstrate over the third substrates closes the microchannels therebyforming capillary channels (e.g., 61 and 62).

In certain embodiments, a microchannel may be formed on a separatesubstrate which may be disposed on to the first substrate on which themicrofluidics array of electrodes is disposed. For example, anothersubstrate may be bonded onto the second area on the first substrate inwhich the microfluidics electrode array is disposed in the first area.The substrate may have a pre-formed microchannel or a microchannel maybe formed after the bonding step. Another substrate with a secondmicrochannel may be disposed on top of the substrate containing themicrochannel to provide an integrated device as depicted in FIG. 2C-2H.The nanopore layer may be disposed on either microchannel at thelocation of intersection of the two microchannels 61 and 62.

In some embodiments, a microchannel may be introduced in the second areaadjacent the first area on the first substrate on which themicrofluidics array of electrodes is disposed. For example, themicrochannel may be etched on the top surface in the second area. Ananopore layer may be placed at a location on the microchannel. Thenanopore layer may include preformed nanopore(s). In alternativeembodiments, nanopore(s) may be formed after positioning the layer at alocation on the microchannel. A third substrate may be prepared byintroducing a microchannel on a bottom surface of third substrate. Thethird substrate may be positioned over the second area on the firstsubstrate such that the top surface of the second area of the firstsubstrate is in contact across its top surface with the bottom surfaceof the third substrate thereby creating closed capillary channels 61 and62.

FIGS. 2I-2K depict devices in which the digital microfluidics module 250and nanopore module 260 share a common bottom (first) substrate 210 onwhich the array of electrodes 249 (a series of individually controllableelectrodes) for the microfluidics module is disposed on a first area anda microfluidic channel 261 is formed in a second area. The microfluidicchannel 261 in the first substrate is aligned with the transferelectrode 271. A second substrate 220 having a single continuouselectrode 255 (e.g., a reference electrode) is disposed in a spacedapart manner from the array of electrodes 249 in the digitalmicrofluidics module 250. A third substrate 230 comprising amicrofluidic channel 262 formed in a lower surface of the thirdsubstrate is placed over the second area of the first surface 210thereby covering the top surface of the first substrate in which themicrofluidic channel 261 is formed. The first substrate and the thirdsubstrate in the nanopore module enclose the microfluidic channels 261and 262 thereby providing capillary channels 261 and 262. It isunderstood that “microfluidic channel(s)” and “microchannel(s)” are usedherein interchangeably to refer to a passage or a cut out in a surfaceof a substrate. Upon placement of a substrate over the passage, thepassage is enclosed forming a capillary channel. Similar to the FIG. 2C,the capillary channels may be fluidically connected to the microfluidicsmodule at one end at the interface 100 between the microfluidics module250 and the nanopore module 260 and with a reservoir or vent on theother end. In other embodiments, the second capillary channel 262 may beconfigured similarly to the capillary channel 62 in FIG. 2H, i.e., thesecond capillary channel 262 may not be connected to the microfluidicsmodule at either end and may be connected to a reservoir/vent at bothends. A top view of the device is depicted in FIG. 2I and a front viewof a cross-section of the device at the interface between the modules isdepicted in FIG. 2I (continued). As is evident from the front view, thedroplet 265 a is on a plane higher than the entrance to the capillarychannel 261. In order to allow the droplet 265 a to flow into thecapillary channel 261, a notch 280 is created in a side edge of thethird substrate 230 to provide space for movement of the droplet downinto the microchannel 261. Thus, the fluidic connection between themicrofluidics module and the nanopore module is provided by a verticalport formed by the notch 280 providing an opening in a top part of thefirst capillary channel 261 at one end of the first capillary channel261 at the interface 100. It is understood that the notch 280 is FIG. 2Iis not drawn to size and may be of any suitable size that allows forfluid communication between the transfer electrode 271 and the firstcapillary channel 261 at the interface 100. Further, the notch may bevaried in size. For example, the notch may be a cut-out that extendsalong a length of the side edge of the third substrate 230 at interface100 and may be proportioned to match the width of the transfer electrode271 or the width of the capillary channel 261 or a length in between.The cut-out may be extended nominally along the width of third substrate230 such that a relatively minor region of the capillary channel 261 isuncovered. In other embodiments, the cut-out may extend over asubstantial length of the capillary channel 261. A layer 270 containinga nanopore is positioned across the first capillary channel 261 at theposition at which the two capillary channels intersect. The layer 270 ispositioned in a support substrate 275. In certain cases, the firstsubstrate 210 may be a glass substrate and the support substrate 275 maybe a PDMS gasket.

A side view of a cross-section of the device shown in FIG. 2I isdepicted in FIG. 2J. The cross-section is at the region of the devicewhere the first capillary channel 261 is aligned with the first transferelectrode 271. Also depicted is a portion of the microfluidics module250 with the array of electrodes 249, the second substrate 220 with asingle electrode 255 (e.g., reference electrode) positioned in a spacedapart manner from the array of electrodes 249. As shown in FIG. 2J, thesingle electrode 255 does not cover the transfer electrodes. While notillustrated in these Figures, the second substrate 220 and the singleelectrode 255 (which may be a reference electrode) may cover thetransfer electrodes 271 and 272, providing a bi-planar electrodeconfiguration. In this embodiment, droplets can be moved to the transferelectrodes 271 and 272 using the bi-planar electrodes. The firstcapillary 261 is located in the first substrate 210 and is located in aplane lower than the plane on which the droplet 265 a is present. Thethird substrate 230 which includes the second microchannel (which isenclosed by the top surface of first substrate 210 to provide thecapillary channel 262) is disposed over the first substrate. The thirdsubstrate 230 includes the notch 280 (or cut out) at the side edgeadjacent to the microfluidics module at the interface 100. The notch 280opens the capillary 261 on a top portion at the end of the capillarychannel 261 providing a vertical port for entrance to the capillarychannel 261. As shown by the direction of the arrow, the droplet travelsdown to the capillary 261 and then proceeds to flow towards theintersection of the first and the second capillary channels. The secondcapillary channel 262 intersects with the first capillary 261 at thelocation of the nanopore layer 270. A support substrate 275 positionedover the first capillary channel 261 (and under the second capillarychannel 262) is depicted. The support substrate 275 includes thenanopore layer 270. As shown in a top view of the nanopore layer isshown in the inset, the support substrate 275 surrounds the nanoporelayer. In some embodiments, the support substrate may be a first layerwith a cut out in the center and a second layer with a cut out in thecenter. The nanopore layer may be disposed at the cut out in between thefirst and the second layers. A nanopore layer in a support substrate maybe used in devices where the bottom substrate 210 is made of glass.

FIG. 2K shows an additional side view of a cross-section of the deviceshown in FIG. 2I. In FIG. 2J, the cross section is at the location ofthe first transfer electrode 271. In FIG. 2K, the cross section is atthe location of the second transfer electrode 272. As shown in FIG. 2K,the entrance to the second capillary channel 262 is aligned with theposition of the droplet 265 b present on the second transfer electrode272. Also depicted in FIG. 2K is the first capillary channel 261 whichintersects with the second capillary channel 262 at the location of thenanopore layer 270.

In another embodiment, as shown in FIG. 2L, the first substrate 210 maybe include a first portion 210 a on which the array of electrodes 249and transfer electrodes 271 and 272 are disposed and a second portion210 b on which a substrate 290 containing capillary channel 261 a isdisposed. Similar to the device shown in FIG. 2I-2K, the capillarychannel 261 a is below the plane on which the transfer electrodes arelocated. Capillary channel 262 is located in substrate 230 where theentrance to the capillary channel 262 is at the same plane as thetransfer electrodes in the microfluidics module 250. Further, similar toFIGS. 2I-2K, entrance to the capillary channel 262 is aligned with thetransfer electrode 272. Thus, a droplet positioned on electrode 272 cantravel substantially horizontally to the capillary channel 262. Similarto the device shown in FIGS. 2I-2K, the substrate 230 includes a notch280 in a side edge of substrate 230 to provide space for a dropletpositioned on transfer electrode 271 to travel down to capillary 261 awhich is located in substrate 290. Also depicted in FIG. 2L is thenanopore layer 270. In this embodiment, the nanopore layer is directlydisposed on the substrate 290 in absence of the support layer 275. Forexample, in embodiments where both substrates containing the channelsare formed from PDMS, the nanopore layer may be directly disposed inbetween the substrates in absence of a support substrate. FIG. 2L, toppanel depicts a side view of a cross section through the device at thelocation at which the transfer electrode 271 and capillary channel 261 aare located. FIG. 2L, bottom panel depicts a side view of a crosssection through the device at the location at which the transferelectrode 272 and the capillary channel 262 are located. From the top,the device looks same as the device shown in FIG. 2I. Thus, the transferelectrodes 271 and 272 are spaced apart same as the transfer electrodes71 and 72 in the device shown in FIG. 2I.

The electrodes in the nanopore module for the transport of moleculesacross the nanopore layer via nanopore(s) may be fabricated afterpositioning of the nanopore layer in the device. For example, theelectrodes may be disposed in openings introduced into the substratesand positioned in the capillary channels such that they are exposed inthe capillary channels and will be in contact with the fluid present inthe capillary channels. The distance of the electrodes from the nanoporemay be determined empirically based on resistance, width, diameter,and/or length of the capillary channel(s).

The nanopore layer may be disposed on either channel. The nanopore layermay be adhered to the surface of the substrate containing themicrochannel by plasma bonding or via a compressible element, such as agasket. In certain cases, the substrate containing the first channel maybe a glass substrate. In this embodiment, a support substrate, such as,a PDMS layer may be used for positioning the nanopore layer. Forexample, the nanopore layer may be provided with a PDMS gasket.

Any suitable method may be employed to form the channels on thesubstrate. In certain cases, lithography or embossing may be used tocreate the channels for the nanopore module. In other embodiments, thechannels may be etched into the substrates. In certain embodiments, acombination of suitable methods may be used to form channels in thesubstrates. For example, a channel may be formed in a glass substrateusing an etching process and another channel may be formed in a PDMSsubstrate using an appropriate method, such as, soft lithography,nanoimprint lithography, laser ablation or embossing (e.g., softembossing). The height/width/diameter of the microchannels may bedetermined empirically. The height/width/diameter of the microchannelsmay be in the range of 0.5 μm to about 50 μm, e.g., 0.5 μm-40 μm-30 2μm-20 3 μm-10 μm, 5 μm-10 μm, such as, 0.5 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, or 50 μm. Asnoted herein, the height/width/diameter of the channels may vary alongthe length of the channels.

In certain embodiments, the nanopore layer (e.g., 70 or 270) may includea coating of insulating material on one or both sides of the nanoporelayer. The insulating material may reduce contact capacitance anddecrease noise associated with detection of translocation of a moleculethrough the nanopore(s) in the nanopore layer. In another embodiment,the surface area of the nanopore layer exposed to a fluid in thecapillary channels in fluid contact with the nanopore layer (e.g.,capillary channels 61 and 62 or 261 and 262) may be reduced. Reducingthe surface area of the nanopore layer that is in contact with a fluidcontaining the molecules to be detected or counted by the nanopore(s)may minimize contact capacitance and reduce background noise. Thesurface area of the nanopore layer in contact with a fluid in thecapillary channels may be reduced by reducing the size of the capillarychannel at the location of the nanopore layer. For example, the heightor width or both (e.g., diameter) of the capillary channels at thelocation of the nanopore layer may be reduced. In another embodiment,the surface area of the nanopore layer may be reduced. In certaindevices, a combination of these embodiments for reducing contactcapacitance may be included. For example, in certain embodiments, anintegrated device as disclosed herein may include capillary channelsthat have a decreased dimension at the location of the nanopore layerand/or may include a nanopore layer that is coated with an insulatingmaterial (e.g., PDMS) on one or both sides of the nanopore layer and/ormay include a nanopore layer having a minimal surface area.

FIG. 3 illustrates another exemplary integrated device which includes amicrofluidics module 300 and a nanopore module 325. In contrast to thenanopore module in FIGS. 1A, 1B, 2A and 2B, the nanopore module 325 isnot functional as a standalone device but functions as a nanopore onceintegrated with the microfluidics module 300. The microfluidics module300 includes an opening 302 sized to allow insertion of the nanoporemodule 325. As depicted in FIG. 3, the microfluidics module includes afluidic droplet 301 that is to be analyzed using the nanopore module325, which contains a layer 311 with a nanopore 305. Upon insertion ofthe nanopore module 325 into the microfluidics module 300, a firstchamber 306 and a second chamber 307 separated by the layer 311 arecreated. The layer 311 also splits the fluid droplet 301 across thenanopore 305.

FIG. 4 provides an integrated device 400 in which the digitalmicrofluidics modules includes a built-in nanopore module. In FIG. 4,the nanopore module is positioned downstream from the area in themicrofluidics module where a fluidic droplet 401 is generated. Themicrofluidics module moves the droplet 401 to the nanopore module suchthat the droplet 401 is split across the layer 402 and is positioned atthe nanopore 403. FIG. 4 shows a top view of the device. The topsubstrate has not been shown for clarity. The nanopore 403 in thenanopore layer 402 has been depicted, although from the top view,nanopore 403 will not be visible. The nanopore layer 402 can be attachedto either the bottom substrate or the top substrate.

In FIGS. 1A, 1B, 2A, 2B, 3 and 4, although a single nanopore is shown,it is understood that the layer may include one or more nanopores. Inaddition, more than one droplet may be positioned in the nanopore moduleor device. The droplet(s) may be analyzed by applying a voltage acrossthe nanopore(s). Applying the voltage may result in movement of chargedmolecules across the nanopore(s). When a tag translocates through thenanopore(s), a decrease in electrical current across the nanoporeprovides an indication of the translocation. In certain embodiments, thechambers of the nanopore module may not be filled with a conductivesolution (e.g., buffer)—the conductive solution may be provided by thefluid droplet once it is positioned across the nanopore layer. Incertain cases, the first and second chambers, across which voltage isapplied for measuring translocation of a tag/aptamer present in thefluid droplet, may be defined by the walls of the nanopore device andthe nanopore layer (e.g., see FIGS. 1B and 2B). The first and secondchambers may be empty prior to the introduction of the fluid droplet ormay contain a conductive fluid. In other cases, the first and secondchambers may be defined walls of the microfluidics module and thenanopore layer (e.g., see FIG. 3). In other cases, the first and secondchambers may be defined by the fluidic droplet split across the nanoporelayer (e.g., see FIGS. 1A, 2A, and 4). In certain cases, the voltage forconducting charged molecules across the nanopore(s) may be applied tothe fluid droplet, for example, in embodiments where a conductivesolution is not present in the chambers. Voltage may be applied to thefluid droplet via electrodes that are in direct or indirect contact withthe fluid droplet. It is understood that the dimension of the nanoporelayer is larger than that of the droplet such that droplet is splitacross the layer and connected only via the nanopore(s).

FIGS. 5A, 5B, 6 and 7 illustrate movement of droplets in devices thathave a digital microfluidics module and a nanopore layer. In FIG. 5A,components of an integrated digital microfluidics/nanopore device 450are depicted. A top view shows that a droplet 401 that is to be analyzedusing the nanopore 403 in the nanopore layer 402 is positioned acrossthe nanopore layer 402. The nanopore 403 is shown here for illustrationpurposes, although from a top view, the nanopore is not visible. Thedevice 450 includes a substrate 411 on which an array of electrodes 405is disposed. The array of electrodes is used to position a droplet 401by splitting the droplet across nanopore layer 402. Arrows 451 and 452depict the direction in which the droplet 401 may be moved across thearray of electrodes to the nanopore layer 402. Upon positioning of thedroplet 401 across the nanopore layer 402, the electrodes 404 and 406positioned below the droplet 401 may be activated to provide adifferential voltage across the nanopore layer 402, thereby facilitatingmovement of molecules (e.g., cleaved tag or aptamer) in the droplet 401across the nanopore 403. The electrodes 404 and 406 are dual functionelectrodes, they serve to move the droplet to the nanopore layer and todrive the tag/aptamer across the nanopore 403.

FIG. 5B depicts a side view of the device 450, a top substrate 412omitted in the top view shown in FIG. 5A is depicted here. The topsubstrate 412 is shown to include an electrode 414. Electrode 414 may bea single electrode or an electrode array. The nanopore layer extendsfrom the top substrate to the bottom substrate. The droplet 401 is splitacross the nanopore layer 402. Although bi-planar electrodes aredepicted in FIG. 5B, the device may not include electrodes in bothsubstrates; rather the top or the bottom substrate may include co-planarelectrodes. The electrodes 404 and 406 in the vicinity of the droplet401 have opposite polarity and drive the tag/aptamer across the nanopore403.

FIG. 6 shows the splitting of droplet 401 across nanopore layer 402having a nanopore 403. 1 a depicts the droplet being moved by theelectrodes 405 in the direction indicated by the arrows towards thenanopore layer 402. In 2 a, the droplet 401 has been split by thenanopore layer 402 and positioned such that the droplet is connected viathe nanopore 403. In 3 a, the electrodes positioned across the nanoporelayer 402 below the droplet 401, are activated to provide an anode (−)and cathode (+). The activated electrodes drive the negatively chargedmolecules (including the tags/aptamers being counted) present in thedroplet 401 through the nanopore 403. As the tags/aptamers translocatethrough the nanopore 403, the number of tags/aptamers may be counted asexplained herein. Step 3 a serves to collect all the tags/aptamers thatwere divided across the nanopore layer, when the droplet was split, inone side of the droplet.

Once substantially all the tags/aptamers have been translocated to oneside of the nanopore membrane, the polarity of the electrodes may bereversed, as shown in 4 a, and the tags/aptamers translocated to theother side of the nanopore layer 402 and counted. The number of tagscounted in step 3 a should be approximately half of the count obtainedin step 4 a. The steps of reversing polarity of electrodes and countingthe tags/aptamers may be repeated any number of times to obtain multiplereadings of the number of tags/aptamers in the droplet.

FIG. 7, 1 b and 2 b show two droplets 600 a and 600 b being moved to thenanopore layer 604 in the directions indicated by the arrows. Once atthe nanopore layer 604, the droplets wet the nanopore layer and arefluidically connected via the nanopore 605 (3 b). In step 4 b, anelectrode positioned below the droplet 600 a is activated to serve as acathode and an electrode positioned below the droplet 600 b is activatedto serve as an anode and the negatively charged cleaved tags/dissociatedaptamers are driven to the droplet 600 a and counted. In step 5 b, thepolarity of the electrodes is reversed and the negatively chargedcleaved tags/dissociated aptamers present in droplet 600 a are driven todroplet 600 b and counted. The steps of reversing polarity of electrodesand counting the cleaved tags/dissociated aptamers may be repeated anynumber of times to obtain multiple readings of the number of cleavedtags/dissociated aptamers in the droplet. The two droplets 600 a and 600b may both be sample droplets (e.g., droplets containing molecules to becounted) or buffer droplets (e.g., for wetting the nanolayer, prior topositioning a sample droplet(s) at the nanopore. In some embodiments,one of the droplets may be a buffer droplet while the other droplet maybe the sample droplet. The tags/aptamers may be counted once or multipletimes.

FIG. 8 illustrates an integrated digital microfluidics and nanoporedevice from a side view. Substrates 91 and 92 are positioned in a spacedapart manner. Substrate 92 includes an electrode 97 and substrate 91includes an electrode array 95. Support structure 98 attaches thenanopore layer 94 to substrate 92. In other embodiments, supportstructure 98 may be attached to the bottom substrate 91. Electrode array95 is used for moving the droplet 99 to the nanopore layer 94, where thenanopore layer splits the droplet and fluidically connects the two sidesof the droplet via the nanopore 93. Electrodes 96 and 97 serve to drivetags/aptamers in the droplet 99 through the nanopore 93. As noted above,the polarity of electrodes 96 and 97 may be reversed to translocate thetags/aptamers through the nanopore a number of times.

Although the figures depict a single nanopore, it is understood thatmore than one nanopore may be present in the nanopore layer. Theelectrodes that flank the nanopore layer and are used to provide avoltage difference across the nanopore layer may or may not be in directcontact with a droplet positioned at the nanopore layer.

The movement of fluidic droplet in the microfluidics and nanoporedevices, modules, and the integrated devices may be carried out via anysuitable means. The means for moving a fluidic droplet in differentdevices/modules and channels, if applicable, may be same or different.For example, fluidic droplets may be moved in the microfluidics deviceor module using fluidic manipulation force, such as, electrowetting,dielectrophoresis, opto-electrowetting, electrode-mediated,electric-field mediated, electrostatic actuation, and the like or acombination thereof. Movement of a fluidic droplet from a microfluidicsmodule to a nanopore module through a fluidic connection, such as, achannel, may be via diffusion, Brownian motion, convection, pumping,applied pressure, gravity-driven flow, density gradients, temperaturegradients, chemical gradients, pressure gradients (positive ornegative), pneumatic pressure, gas-producing chemical reactions,centrifugal flow, capillary pressure, wicking, electric field-mediated,electrode-mediated, electrophoresis, dielectrophoresis, magnetophoresis,magnetic fields, magnetically driven flow, optical force, chemotaxis,phototaxis, surface tension gradient driven flow, Marangoni stresses,thermo-capillary convection, surface energy gradients, acoustophoresis,surface acoustic waves, electroosmotic flow, thermophoresis,electrowetting, opto-electrowetting, or combinations thereof. A fluidicdroplet may be moved in the nanopore module and positioned across thenanopore layer via fluidic manipulation force, such as, electrowetting,dielectrophoresis, opto-electrowetting, electrode-mediated,electric-field mediated, electrostatic actuation, and the like or acombination thereof. The tag/aptamer in the droplet may be translocatedthrough the nanopore(s) using electric potential, electrostaticpotential, electrokinetic flow, electro-osmotic flow, pressure-inducedflow, electrophoresis, electrophoretic transport, electro-osmotictransport, diffusion transport, electric-field mediated flow,dielectrophoretic mediated transport of the tag/aptamer, and othermethods known to skill in the art or combinations thereof.

Exemplary embodiments of the present disclosure include counting thenumber of tags present in the droplet positioned across the nanoporelayer by first translocating substantially all tags to the same side ofthe nanopore layer to collect all the tags in a cis or trans chamber,followed by translocating the tags to the other side of the nanoporelayer and counting the number of tags traversing through the nanopore(s)in the nanopore layer. As used herein, “cis” and “trans” in the contextof a nanopore layer refers to the opposite sides of the nanopore layer.These terms are used to in context of a side of the nanopore layer andalso in the context of a chamber on a side of the nanopore layer. As isunderstood from the description of the devices, the cis and transchambers may be defined by physical structures defined by walls,substrates, etc. In some cases, the cis and trans chambers may bedefined by a droplet placed across a nanopore layer. The droplet may bein contact with a wall or substrate on one or more sides of the droplet.In certain cases, cis and trans chambers may be defined by the droplet,the cis chamber may extend from the cis side of the nanopore layer tothe periphery of the portion of the droplet on the cis side and thetrans chamber may extend from the trans side of the nanopore layer tothe periphery of the portion of the droplet on the trans side. A portionof the droplet on each of cis and trans side may be in contact with asubstrate. Thus, the cis and trans chamber may be defined by acombination of the periphery of the droplet, a portion of the substratesand the nanopore layer.

In certain cases, the microfluidics device and/or the microfluidicsmodule may include an inert fluid that is immiscible with the sampledroplet and the reagent droplets. For example, the inert fluid may be aheavy fluid that is denser than water, such as oil that is immisciblewith the fluidic droplets being generated and processed in themicrofluidics module. The inert fluid may facilitate formation of thefluidic droplets as well as increase stability of the shape of the fluiddroplets and may further be useful for keeping the different dropletsspatially separated from one another. Exemplary inert fluids includepolar liquids, silicone oil, fluorosilicone oil, hydrocarbons, alkanes,mineral oil, and paraffin oil. In certain cases, the microfluidicsdevice or module and the inert fluid may be as disclosed inUS20070242105, which is herein incorporated by reference in itsentirety. In other embodiments, an immiscible fluid is not included inthe device. In these embodiments, the ambient air fills the spaces inthe device.

As used herein, “droplet(s)” and “fluidic droplet(s)” are usedinterchangeably to refer to a discreet volume of liquid that is roughlyspherical in shape and is bounded on at least two sides by a wall orsubstrate of the microfluidics device, the nanopore device,microfluidics module, or the nanopore module. Roughly spherical in thecontext of the droplet refers to shapes such as spherical, partiallyflattened sphere, e.g., disc shaped, slug shaped, truncated sphere,ellipsoid, hemispherical, or ovoid. The volume of the droplet in themicrofluidics and nanopore modules and devices disclosed herein mayrange from about 10 μL to about 5 pL, such as, 10 μL-1 pL, 7.5 μL-10 pL,5 μL-1 nL, 2.5 μL-10 nL, or 1 μL-100 nL, e.g., 10 μL, 1 μL, 800 nL, 400nL, 100 nL, 10 nL, or lesser.

In certain embodiments, the integrated device may include amicrofluidics module with a built-in nanopore module. The integrateddevice may include a first substrate and a second substrate with a gapseparating the first and second substrates, the gap (which may be filledwith air or an immiscible liquid) providing the space in which a sampledroplet is contacted with the first binding member (either immobilizedon a magnetic bead or on one of the two substrates); optionally awashing step is performed; followed by contacting the analyte bound tothe first binding member with the second binding member; optional mixingand wash step may be performed; and the tag attached to the secondbinding member is cleaved to generate a droplet containing the cleavedtag. The droplet containing the cleaved tag may then be positionedacross a nanopore layer located in the gap between the first and secondsubstrates.

As noted herein, the droplets may be moved in the integrated device vianumerous ways, such as, using a programmable fluidic manipulation force(e.g., electrowetting, dielectrophoresis, electrostatic actuation,electric field-mediated, electrode-mediated force, SAW, etc.). Incertain cases, the microfluidics device and module may move droplets ofsample and reagents for conducting analyte analysis by using electrodes.The electrodes may be co-planar, i.e., present on the same substrate orin a facing orientation (bi-planar), i.e., present in the first andsecond substrates. In certain cases, the microfluidics device or modulemay have the electrode configurations as described in U.S. Pat. No.6,911,132, which is herein incorporated by reference in its entirety. Incertain cases, the device may include a first substrate separated from asecond substrate by a gap; the first substrate may include a series ofelectrodes positioned on an upper surface; a dielectric layer may bedisposed on the upper surface of the first substrate and covering theseries of electrodes to provide a substantially planar surface formovement of the droplets. Optionally, a layer of hydrophobic materialmay be placed on the upper surface of the dielectric layer to provide asubstantially planar surface. In certain cases, the first substrate mayinclude co-planar electrodes—e.g., drive/control and referenceelectrodes present on a single substrate. In other cases, the secondsubstrate that is positioned over the first substrate may include anelectrode on lower surface of the second substrate, where the lowersurface of the second substrate is facing the upper surface of the firstsubstrate. The electrode on the second substrates may be covered with aninsulating material. The series of electrodes may be arranged in alongitudinal direction along a length of the microfluidics module or ina lateral direction along a width of the microfluidics module or both(e.g., a two-dimensional array or grid). In certain cases, the array ofelectrodes may be activated (e.g., turned on and off) by a processor ofa computer operably coupled to the device for moving the droplets in aprogrammable manner. Devices and methods for actuating droplets in amicrofluidics device are known. In exemplary cases, the microfluidicsmodule may be similar to a droplet actuator known in the field. Forexample, the first (bottom) substrate may contain a patterned array ofindividually controllable electrodes, and the second (top) substrate mayinclude a continuous grounding electrode. A dielectric insulator coatedwith a hydrophobic may be coated over the electrodes to decrease thewettability of the surface and to add capacitance between the dropletand the control electrodes (the patterned array of electrodes). In orderto move a droplet, a control voltage may be applied to an electrode (inthe array of electrodes) adjacent to the droplet, and at the same time,the electrode just under the droplet is deactivated. By varying theelectric potential along a linear array of electrodes, electrowettingcan be used to move droplets along this line of electrodes.

The first and second substrates may be made from any suitable material.Suitable materials without limitation include paper, thin film polymer,silica, silicon, processed silicon, glass (rigid or flexible), polymers(rigid, flexible, opaque, or transparent) (e.g., polymethylmethacrylate(PMMA) and cyclic olefin copolymer (COC), polystyrene (PS),polycarbonate (PC), printed circuit board, and polydimethylsiloxane(PDMS). In certain cases, at least the first or the second substrate maybe substantially transparent. Substantially transparent substrate may beused in devices where photocleavage of tag attached to a second bindingmember is performed. In embodiments, where co-planar electrodes arepresent in one of the substrates, the electrodes may or may not betransparent. In other embodiments, such as, where electrodes are infacing orientation, (present in both substrates) the electrodes on atleast one of the substrates may be substantially transparent, forexample, the electrodes may be made from indium tin oxide. Theelectrodes may be made of any suitable material. The electrodes may bemade of any conductive material such as pure metals or alloys, or otherconductive materials. Examples include aluminum, carbon (such asgraphite), chromium, cobalt, copper, gallium, gold, indium, iridium,iron, lead, magnesium, mercury (as an amalgam), nickel, niobium, osmium,palladium, platinum, rhenium, rhodium, selenium, silicon (such as highlydoped polycrystalline silicon), silver, tantalum, tin, titanium,tungsten, vanadium, zinc, zirconium, mixtures thereof, and alloys ormetallic compounds of these elements. In certain embodiments, theconductive material includes carbon, gold, platinum, palladium, iridium,or alloys of these metals, since such noble metals and their alloys areunreactive in aqueous environment.

In certain cases, the first substrate or the second substrate may have afirst binding member immobilized thereon in the gap. For example, asurface of the first substrate that is in facing relationship to asurface of the second substrate may include an area on which a firstbinding member is disposed. As noted herein, the first binding member(e.g., a polypeptide, for example, a receptor, an antibody or afunctional fragment thereof) may be immobilized on the surface of asolid substrate using any conventional method. In certain cases, a firstposition on the surface of the first or the second substrate in the gapmay only include one type of binding member (e.g., a single type ofantibody). In other embodiments, a first position on the surface of thefirst or the second substrate in the gap may only include a plurality ofdifferent binding members, for analysis of multiple analytes.Alternatively, the device may include a plurality of locations on thesurface of the first or second substrates where each location mayinclude a different first binding member immobilized thereupon.

In embodiments where a surface of the first substrate or the secondsubstrate in the gap has a plurality of locations at which differentfirst binding members are immobilized, the locations may be arrangedlinearly along a length of the device. A sample droplet may be movedlinearly to sequentially contact each of the plurality of the locations.In another embodiment, a sample may be split into multiple droplets andeach of the droplets may independently contact the each of the pluralityof the locations. As noted herein, the first binding member may not beattached to the first or the second substrate and may be attached to abead that may be introduced in the microfluidics device as, e.g., adroplet.

As noted herein, a sample and any reagents for assaying the sample maybe manipulated as discrete volumes of fluid that may be moved in betweenthe first and second substrates using a programmable fluidicmanipulation force (e.g., electrowetting, dielectrophoresis,electrostatic actuation, electric field-mediated, electrode-mediatedforce, etc.). For example, at least one of the first and secondsubstrates may include an array of electrodes for manipulating discretevolumes of fluid, e.g., moving droplets from one location to another inbetween the first and second substrates, mixing, merging splitting,diluting, etc. In another example, surface acoustic waves may be used tomove droplets for the analyte analysis method.

In another embodiment, the microfluidics module may move droplets ofsample and reagents for conducting analyte analysis by using surfaceacoustics waves. In these embodiments the first substrate may a thinplanar material conducive to propagation of surface acoustic waves. Thefirst substrate may be a piezoelectric crystal layer, such a lithiumniobate (LiNbO₃), quartz, LiTaO₃ wafer. In certain cases, thepiezoelectric wafer may be removably coupled to a supersubstrate, wheresurface acoustic waves (SAWs) generated from a transducer is transmittedto the supersubstrate via a coupling medium disposed between thepiezoelectric crystal layer and the supersubstrate. The upper surface ofthe supersubstrate may be overlayed by a second substrate and a dropletmay be moved in a space between the second substrate and upper surfaceof the supersubstrate via SAWs generated by an interdigitated transducerconnected to the piezoelectric crystal layer. In certain cases, themicrofluidics module may be a SAW microfluidics device described inWO2011/023949, which is herein incorporated by reference.

In an alternate embodiment, the microfluidics module may include a firstsurface separated from a second surface with a space between the firstsurface and the second surface, where sample and reagent droplets aremanipulated for performing the sample analysis disclosed herein. Themicrofluidics device may further include a layer of surface acousticwave (SAW) generation material coupled to the first surface; and atransducer electrode structure arranged at the SAW generation materiallayer to provide surface acoustic waves (SAWs) at the first surface fortransmission to droplets on the first surface, where the first surfacehas at least one SAW scattering element for affecting the transmission,distribution and/or behavior of SAWs at the first surface, and where theSAW generation material is selected from the group consisting of:polycrystalline material, textured polycrystalline material, biaxiallytextured polycrystalline material, microcrystalline material,nanocrystalline material, amorphous material and composite material. Incertain cases, the SAW generation material may be ferroelectricmaterial, pyroelectric material, piezoelectric material ormagnetostrictive material. The arrangement of the SAW scatteringelements may provide, in effect, a phononic crystal structure thatinteracts with or affects the acoustic field at the first surface toaffect movement of droplet on the first surface. In certain cases, themicrofluidics module may be a SAW microfluidics device described inUS20130330247, which is herein incorporated by reference. The SAWmicrofluidics device may be used in conjunction with a nanopore deviceor may have a nanopore module integrated therewith.

The devices described herein may be used in conjunction with anotherdevice or devices, such as, a power source, an acoustic wave generator,and the like.

The device that may be used for carrying out the method steps describedherein may also include means for supplying reagent and collecting wastematerials. Such means may include chambers, absorption pads, reservoirs,etc. These means may be fluidically connected to the device.

The microfluidics module may be fluidically connected to reservoirs forsupplying sample analysis reagents, such as, first binding member,second binding member, wash buffer, cleavage inducing reagent and thelike. The nanopore module may be fluidically connected to a reservoirfor collecting waste materials, reservoirs for supplying conductivesolution to the cis and trans chambers and the like.

The integrated device may be automatic or semi-automatic and may beremovably coupled to a housing comprising a source of electricity forsupplying voltage to the electrodes and a random access memory forstoring instructions for contacting the sample with a first bindingmember, wherein the first binding member is immobilized on a solidsupport and wherein the first binding member specifically binds to theanalyte; contacting the analyte with a second binding member, whereinthe second binding member specifically binds to the analyte and whereinthe second binding member comprises a cleavable tag attached thereto;removing second binding member not bound to the analyte bound to thefirst binding member; cleaving the tag attached to the second bindingmember bound to the analyte bound to the first binding member;translocating the tag through or across nanopores in a layer;determining the number of tags translocating through the layer;measuring the analyte in the sample based on the numbers of tagstranslocating through the layer or the time to translocate a knownnumber of tags for a fixed interval of time. As noted herein, theanalyte analysis method may be executed using a processor that controlsthe device. For example, the device may be programed to perform analyteanalysis as disclosed herein, including any optional mixing, incubating,and washing steps as disclosed herein. The housing may further include aprocessor for executing the instructions stored in the memory. Thedevices described herein may include a data acquisition module (DAQ) forprocessing electrical signals from the nanopore device or module. Incertain cases, a patch-clamp amplifier for processing electrical signalsand achieving optimal signal to noise ratio may also be included.

In certain cases, the devices described herein may be associated with asystem for automatically performing at least some steps of the analyteanalysis methods. An example of such a system is shown in FIG. 9. TheExemplary system includes a processing component 60 including a dataprocessing unit 63 having a processor and memory, operatively coupled todisplay 61 and a transmitter/receiver unit 62 that is in communication64 with a receiver/transmitter unit 69 of a device 68 of the presentdisclosure. The device 68 is controlled by the processing component 60that executes instructions (steps of a program) to perform at least somesteps of the analyte analysis methods disclosed herein. In certaincases, the processing component 60 may be a computer, a meter with anopening for insertion of the integrated device (the opening may be aslot sized and shaped to accommodate the device and operably connect tothe device), or a combination thereof. The communication 64 between theprocessing component 60 and the device 68 may be wired or wireless. Thedevice 68 may be any device described herein with microfluidics 66 andnanopore 67 functionality. In certain cases, the movement of a dropletin the devices disclosed herein may be programmed as disclosed in U.S.Pat. No. 6,294,063, which is herein incorporated by reference in itsentirety.

The various illustrative processes described in connection with theembodiments herein may be implemented or performed with a generalpurpose processor, a Digital Signal Processor (DSP), an ApplicationSpecific Integrated Circuit (ASIC), a Field Programmable Gate Array(FPGA) or other programmable logic device, discrete gate or transistorlogic, discrete hardware components, or any combination thereof designedto perform the functions described herein. A general purpose processormay be a microprocessor, but in the alternative, the processor may beany conventional processor, controller, microcontroller, or statemachine. The processor can be part of a computing system that also has auser interface port that communicates with a user interface, and whichreceives commands entered by a user, has at least one memory (e.g., harddrive or other comparable storage, and random access memory) that storeselectronic information including a program that operates under controlof the processor and with communication via the user interface port, anda video output that produces its output via any kind of video outputformat, e.g., VGA, DVI, HDMI, DisplayPort, or any other form.

A processor may also be implemented as a combination of computingdevices, e.g., a combination of a DSP and a microprocessor, a pluralityof microprocessors, one or more microprocessors in conjunction with aDSP core, or any other such configuration. These devices may also beused to select values for devices as described herein. The camera may bea camera based on phototubes, photodiodes, active pixel sensors (CMOS),CCD, photoresistors, photovoltaic cells or other digital image capturetechnology.

The steps of a method or algorithm described in connection with theembodiments disclosed herein may be embodied directly in hardware, in asoftware module executed by a processor, or in a combination of the two.A software module may reside in Random Access Memory (RAM), flashmemory, Read Only Memory (ROM), Electrically Programmable ROM (EPROM),Electrically Erasable Programmable ROM (EEPROM), registers, hard disk, aremovable disk, a CD-ROM, a cloud, or any other form of storage mediumknown in the art. An exemplary storage medium is coupled to theprocessor such that the processor can read information from, and writeinformation to, the storage medium. In the alternative, the storagemedium may be integral to the processor. The processor and the storagemedium may reside in an ASIC. The ASIC may reside in a user terminal. Inthe alternative, the processor and the storage medium may reside asdiscrete components in a user terminal.

In one or more example embodiments, the functions described may beimplemented in hardware, software, firmware, or any combination thereof.If implemented in software, the functions may be stored on, transmittedover or resulting analysis/calculation data output as one or moreinstructions, code or other information on a computer-readable medium.Computer-readable media includes both computer storage media andcommunication media including any medium that facilitates transfer of acomputer program from one place to another. A storage media may be anyavailable non-transitory media that can be accessed by a computer. Byway of example, such computer-readable media can include RAM, ROM,EEPROM, CD-ROM or other optical disk storage, magnetic disk storage orother magnetic storage devices, or any other medium that can be used tocarry or store desired program code in the form of instructions or datastructures and that can be accessed by a computer. The memory storagecan also be rotating magnetic hard disk drives, optical disk drives, orflash memory based storage drives or other such solid state, magnetic,or optical storage devices. Disk and disc, as used herein, includescompact disc (CD), laser disc, optical disc, digital versatile disc(DVD), floppy disk and Blu-ray disc where disks usually reproduce datamagnetically, while discs reproduce data optically with lasers.Combinations of the above should also be included within the scope ofcomputer-readable media.

To the extent the embodiments disclosed herein include or operate inassociation with memory, storage, and/or computer readable media, thenthat memory, storage, and/or computer readable media are non-transitory.Accordingly, to the extent that memory, storage, and/or computerreadable media are covered by one or more claims, then that memory,storage, and/or computer readable media is only non-transitory.

In certain cases, the device may be a microfluidic device, such as alab-on-chip device, continuous-flow microfluidic device, ordroplet-based microfluidic device, where analyte analysis may be carriedout in a droplet of the sample containing or suspected of containing ananalyte. Exemplary microfluidic devices that may be used in the presentmethods include those described in WO2007136386, U.S. Pat. No.8,287,808, WO2009111431, WO2010040227, WO2011137533, WO2013066441,WO2014062551, or WO2014066704. In certain cases, the device may bedigital microfluidics device (DMF), a surface acoustic wave basedmicrofluidic device (SAW), a fully integrated DMF and nanopore device,or a fully integrated SAW and nanopore device. In some embodiments, theDMF element and a nanopore element are operatively coupled in the fullyintegrated DMF and nanopore device, or a SAW element and a nanoporeelement are operatively coupled in the fully integrated SAW and nanoporedevice. In some embodiments, the DMF device or the SAW device isfabricated by roll to roll based printed electronics method. In someembodiments, the DMF element or the SAW element is fabricated by roll toroll based printed electronic methods. In some embodiments, the fullyintegrated DMF and nanopore device or the fully integrated SAW andnanopore device comprise a microfluidic conduit. In some embodiments,the microfluidic conduit couples the DMF element to the nanoporeelement, and the microfluidic conduit comprises a fluidic flow that isinduced by passive forces or active forces.

Exemplary electrowetting techniques can be found in U.S. Pat. No.8,637,242. Electrophoresis on a microscale such as that described inWO2011057197 may be also utilized. An exemplary dielectrophoresistechnique is described in U.S. Pat. No. 6,294,063.

The devices of the present disclosure are generally free of externalpumps and valves and are thus economical to manufacture and use. Thedevices and associated systems disclosed herein, as well as all themethods disclosed herein, are useful for applications in the field, suchas, for analysis of a sample at the source of the sample, such as, atpoint-of-care (e.g., in the clinics, hospitals, physician's office, corelaboratory facility, in home, and the like). In some cases, a device orsystem of the present disclosure (e.g., also as used in the methodsdisclosed herein) includes a heat source or a light source configured toinduce, when the heat source or light source is activated, cleavage of athermally cleavable or a photocleavable linker linking the tag to theanalyte, as described herein.

The present disclosure also describes a microfluidics device used inconjunction with a nanopore-enabled device and an integratedmicrofluidics nanopore-enabled device. A nanopore-enabled device refersto a device which includes a layer or membrane in which a nanopore canbe created. A nanopore-enabled device of the present disclosure includestwo chambers separated by the layer or membrane, where the two chambersinclude an ionic liquid, (e.g., a salt solution, with or without ananalyte of interest) for conducting current. A nanopore may be createdin the layer of the nanopore-enable device by applying a voltage acrossthe layer using the ionic liquid (e.g., salt solution, with or withoutan analyte of interest) in the chambers. As will be understood any ofthe nanopore devices (used in conjunction with a microfluidics device orintegrated with a microfluidics module) described herein may initiallybe provided as a nanopore-enabled device that includes a layer in whicha nanopore can be formed but is devoid of a nanopore. A nanopore may becreated in the nanopore-enabled device during use, such as, prior tousing the nanopore for detecting translocation of a tag. In certainembodiments, an ionic liquid, e.g., salt solution, containing the tag tobe detected by the nanopore may be used for both creating the nanoporeand for translocating a tag across the created nanopore.

In some embodiments, a quality of the nanopore that is created byapplying voltage across the layer, as described above, is assessed bythe level of noise in a current measured when a baseline voltage isapplied across the nanopore layer or membrane.

In some cases, the nanopore created by applying voltage across thelayer, as described above, may be conditioned to physically alter thenanopore and to obtain a desired electroosmotic property, e.g., increasethe pore size and/or to reduce noise in the measured current across thenanopore when a voltage is applied across the nanopore layer ormembrane. Thus, in some embodiments, a method of generating a nanoporein an integrated digital microfluidics nanopore-enabled device mayinclude conditioning the nanopore. Conditioning may include: alternatelyapplying a first voltage having a first polarity and a second voltagehaving a second polarity opposite the first polarity across the nanoporelayer or membrane, wherein the first and second voltages are eachapplied at least once; and measuring an electroosmotic property relatedto a size of the nanopore. In some cases, the electroosmotic propertyrelated to a size of the nanopore is measured before the conditioning,to obtain an initial estimate of the size of the nanopore.

The electroosmotic property may be any suitable property that providesan estimate for the size of the nanopore. In some cases, theelectroosmotic property is represented by a current-voltage curveobtained over a range of voltages (a range of −1 V to 1 V, e.g., −500 mVto 500 mV, −250 mV to 250 mV, −200 mV to 200 mV, 10 mV to 500 mV, 10 mVto 250 mV, 10 mV to 200 mV, including 15 mV to 200 mV). In some cases,the electroosmotic property is a conductance or resistance measuredacross the nanopore layer or membrane.

The first and second voltage may have any suitable magnitude formodifying the nanopore and to obtain the desired electroosmoticproperties. In some cases, the first and second voltages have amagnitude or 100 mV or more, e.g., 200 mV or more, 500 mV or more, 750mV or more, 1.0 V or more, 2.0 V of more, 3.0 V or more, including 4.0 Vor more, and in some cases has a magnitude of 10 V or less, e.g., 9.0 Vor less, 8.0 V or less, 6.0 V or less, including 4.0 V or less. In someembodiments, the first and second voltages have a magnitude in the rangeof 100 mV to 10 V, e.g., 200 mV to 9.0 V, 250 mV to 9.0 V, 500 mV to 9.0V, 1.0 V to 8.0 V, including 2.0 V to 6.0 V.

The first and second voltages may each be applied for any suitablelength of time for modifying the nanopore and to obtain the desiredelectroosmotic properties. In some cases, the first and second voltagesare each applied for 10 milliseconds (ms) or more, e.g., 100 ms or more,200 ms or more, 500 ms or more, 1 second (s) or more, 2 s or more,including 3 s or more, and in some cases, is applied for 10 s or less,e.g, 5 s or less, 4 s or less, 3 s or less, 2 s or less, 1 s or less,500 ms or less, 200 ms or less, including 100 ms or less. In some cases,the first and second voltages are each applied for a duration in therange of 10 ms to 100 ms, 100 ms to 200 ms, 200 ms to 500 ms, 500 ms to1 s, 1 s to 2 s, 2 s to 3 s, 3 s to 4 s, 3 s to 5 s, or 3 s to 10 s.

The first and second voltages may each be applied any suitable number oftimes for modifying the nanopore and to obtain the desiredelectroosmotic properties. In some cases, the first and second voltagesare each applied twice or more, three times or more, 4 times or more, 5times or more, 7 times or more 10 times or more, 20 times or more, 30times or more, 50 times or more, 100 times or more, 200 times or more,including 500 times or more, and in some embodiments, is applied for10,000 time or less, e.g., 5,000 times or less, 1,000 times or less, 500times or less, 400 times or less, 200 times or less, 100 times or less,including 50 times or less. In some embodiments, the first and secondvoltages are each applied from two to 50 times, 10 to 50 times, 30 to 50times, 50 to 100 times, 100 to 200 times, 100 to 500 times, 500 to 1,000times, 500 to 1,000 times, or 500 to 10,000 times.

4. INTEGRATION OF A NANOPORE MODULE ON ONE SIDE OF A DMF MODULE

An aspect of the present disclosure includes an integrated device thatincludes a digital microfluidics (DMF) module and a nanopore layerpositioned on one exterior side of the DMF module (FIG. 40). Thenanopore of the nanopore layer may be accessed by a droplet in aninternal space of the DMF module through a hole (also referred to as an“opening”) that is present in the first (e.g., top) or second (e.g.,bottom) substrate of the DMF module or through a side of the DMF modulebetween the first and second substrate. As described above, the nanoporelayer may include a nanopore membrane or substrate, which in some casesmay be a commercially available silicon nitride (SiN_(x)) membrane in atransmission electron microscope (TEM) window. The nanopore layer formsa seal over the hole such that, in the absence of a nanopore (i.e. priorto fabrication of a nanopore, as described herein), a volume of liquidin the DMF module is physically isolated from any volume of liquid on oraround the outside of the nanopore layer. In some cases, the nanoporelayer is part of a nanopore module, where the nanopore layer separates acompartment within the nanopore module from a volume of liquid in theDMF module (e.g., a liquid droplet in the hole of the substrate, asdescribed above). The nanopore layer or module is sealed to the outersurface of the substrate such that a volume of liquid (e.g., a liquiddroplet in the hole of the substrate) is physically isolated from theoutside environment.

The hole in the substrate through which a liquid droplet in the DMF hasaccess to the nanopore layer may be dimensioned to be suitable for aliquid droplet to move through the hole by capillary action. Thus, thehole in the substrate may be a capillary channel. The hole may have anysuitable cross-sectional shape and dimensions to support movement of aliquid droplet through the hole passively, e.g., by capillary action. Insome cases, the diameter of the hole is wider on the side of the DMFthan the diameter of the hole on the external side (i.e., the sidefacing the nanopore layer). In some cases, the angle between the bottomsurface of the substrate and the wall of the hole is right angle orobtuse (e.g., 90° or greater, e.g., 95° or greater, including 100° orgreater).

The integrated DMF-nanopore module device may include a pair ofelectrodes, which may find use in fabricating the nanopore in thenanopore layer and/or for detecting an analyte of interest that has beenprocessed by the DMF module, as described elsewhere herein. The pair ofelectrodes may be made of any suitable material, including, but notlimited to, indium tin oxide (ITO). The pair electrodes may beconfigured in any suitable manner. In some embodiments, one electrode ispositioned in a compartment in the nanopore module, and a secondelectrode is positioned in the DMF module, by physically penetrating thesubstrate to access the volume of liquid on the other side of thenanopore layer (FIG. 40).

In some embodiments, the first electrode may be the same electrode asthe single continuous electrode (e.g., the reference electrode) used inthe DMF module, and the second electrode may be disposed on the topsurface (i.e., outer surface) of the substrate opposite the bottomsurface on which the first electrode is positioned (FIG. 43). In suchcases, the top surface may be treated in a similar manner as the bottomsurface (e.g., coating with an electrode material, such as indium tinoxide, and a polymer, such as polytetrafluoroethylene (includingTeflon®). Thus, in some cases, where the second electrode is anelectrode on the top surface of the substrate to which the nanoporelayer/module is attached, the volume of liquid on the outside surface ofthe nanopore layer relative to the DMF module is in electrical contactwith the second electrode. The electrical path for the nanoporefabrication may be represented as: second electrode->liquid(external)->nanopore membrane (without a nanopore)->liquid (internal toDMF module)-> first electrode (same as the single continuous electrodeof the DMF). The second electrode may also be absent from the area wherethe nanopore layer/module is attached so as to force current into theliquid on the outside of the nanopore membrane, which in some cases maybe contained within the nanopore module.

In some embodiments, as shown in FIG. 44, the first electrode is thesame electrode as the single continuous electrode (e.g., the referenceelectrode) used in a first DMF module (e.g., “bottom DMF chip” in FIG.44), and the second electrode may be provided by a second DMF module(e.g., “top DMF chip” in FIG. 44) having a hole in a corresponding topsubstrate associated with the single continuous electrode of the secondDMF module, and the nanopore layer is interposed between the two DMFmodules between the holes in the respective substrates. Thus, the firstand second DMF modules may be reversed in orientation relative to eachother such that the top substrate associated with the single continuouselectrode of the first DMF module is proximal to and faces the topsubstrate associated with the single continuous electrode of the secondDMF module. The two DMF modules may be positioned relative to each othersuch that, when there is a nanopore in the nanopore layer, the two DMFmodules are fluidically and electrically coupled together through thenanopore membrane. Prior to formation of the nanopore, the two volumesof fluid in the two DMF modules may be isolated from each other. In somecases, a structural layer is interposed between the two DMF modules toprovide structural support and reduce bending.

Also provided herein is a method of making a nanopore in ananopore-enabled layer, in an integrated DMF-nanopore module device, asdescribed above. An implementation of the method may include positioningan ionic liquid, e.g., a salt solution (e.g., LiCl, KCl, etc.) to thehole in the DMF module using any suitable method, as described herein,and allowing capillary action to move the liquid through the hole (see,e.g., FIG. 40). An ionic liquid, e.g., a salt solution, may bepositioned on the other side of the nanopore-enabled layer (i.e., thenanopore membrane before making a nanopore) The nanopore module issealed from the DMF module, using any suitable method, such as, but notlimited to PDMS, pressure, wax, adhesive, etc., such that the liquidvolume in the hole is isolated from a liquid volume on the other side ofthe nanopore membrane. Application of an electric field, such as avoltage across the nanopore-enabled layer leads to the eventualformation of a nanopore, which can be readily detected, e.g., as adielectric breakdown in a current trace.

After creation of a nanopore in the nanopore layer, in some cases, aconditioning process may be carried out to physically modify thenanopore and clean the signal. In some cases, the conditioning includesvarying the voltage applied across the nanopore over time.

After nanopore fabrication, the DMF module may be re-activated tocomplete any liquid pre-processing steps for translocation (e.g. replacesolution in the DMF, such as replacing KCl with LiCl). Afterpre-processing, the DMF liquid volume, e.g., a liquid sample containingan analyte of interest, may be positioned in the hole. The DMF systemmay then be de-activated and the nanopore module may be enabled to allowand detect translocation events.

5. VARIATIONS ON METHODS AND ON USE OF THE DEVICE

The disclosed methods of determining the presence or amount of analyteof interest present in a sample, and the use of the microfluidicsdevice, may be as described above. The methods and use of the disclosedmicrofluidics device may also be adapted in view of other methods foranalyzing analytes. Examples of well-known variations include, but arenot limited to, immunoassay, such as sandwich immunoassay (e.g.,monoclonal-polyclonal sandwich immunoassays, including enzyme detection(enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA),competitive inhibition immunoassay (e.g., forward and reverse), enzymemultiplied immunoassay technique (EMIT), a competitive binding assay,bioluminescence resonance energy transfer (BRET), one-step antibodydetection assay, homogeneous assay, heterogeneous assay, capture on thefly assay, etc. In some instances, the descriptions below may overlapthe method described above; in others, the descriptions below mayprovide alternates.

a) Immunoassay

The analyte of interest, and/or peptides or fragments thereof, may beanalyzed using an immunoassay. The presence or amount of analyte ofinterest can be determined using the herein-described antibodies anddetecting specific binding to analyte of interest. Any immunoassay maybe utilized. The immunoassay may be an enzyme-linked immunoassay(ELISA), a competitive inhibition assay, such as forward or reversecompetitive inhibition assays, or a competitive binding assay, forexample. In some embodiments, one tag is attached to the captureantibody and the detection antibody. Alternately, a microparticle ornanoparticle employed for capture, also can function for detection(e.g., where it is attached or associated by some means to a cleavablelinker).

A heterogeneous format may be used. For example, after the test sampleis obtained from a subject, a first mixture is prepared. The mixturecontains the test sample being assessed for analyte of interest and afirst specific binding partner, wherein the first specific bindingpartner and any analyte of interest contained in the test sample form afirst specific binding partner-analyte of interest complex. Preferably,the first specific binding partner is an anti-analyte of interestantibody or a fragment thereof. The order in which the test sample andthe first specific binding partner are added to form the mixture is notcritical. Preferably, the first specific binding partner is immobilizedon a solid phase. The solid phase used in the immunoassay (for the firstspecific binding partner and, optionally, the second specific bindingpartner) can be any solid phase known in the art, such as, but notlimited to, a magnetic particle, a bead a nanobead, a microbead, ananoparticle, a microparticle, a membrane, a scaffolding molecule, afilm, a filter paper, a disc, or a chip (e.g., a microfluidic chip).

After the mixture containing the first specific binding partner-analyteof interest complex is formed, any unbound analyte of interest isremoved from the complex using any technique known in the art. Forexample, the unbound analyte of interest can be removed by washing.Desirably, however, the first specific binding partner is present inexcess of any analyte of interest present in the test sample, such thatall analyte of interest that is present in the test sample is bound bythe first specific binding partner.

After any unbound analyte of interest is removed, a second specificbinding partner is added to the mixture to form a first specific bindingpartner-analyte of interest-second specific binding partner complex. Thesecond specific binding partner is preferably an anti-analyte ofinterest antibody that binds to an epitope on analyte of interest thatdiffers from the epitope on analyte of interest bound by the firstspecific binding partner. Moreover, also preferably, the second specificbinding partner is labeled with or contains a detectable label (e.g.,tag attached by a cleavable linker, as described above).

The use of immobilized antibodies or fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles, latex particles or modified surface latex particles, polymeror polymer film, plastic or plastic film, planar substrate, amicrofluidic surface, pieces of a solid substrate material, and thelike.

b) Sandwich Immunoassay

The sandwich immunoassay measures the amount of antigen between twolayers of antibodies (i.e., a capture antibody (i.e., at least onecapture antibody) and a detection antibody (i.e. at least one detectionantibody). The capture antibody and the detection antibody bind todifferent epitopes on the antigen, e.g., analyte of interest. Desirably,binding of the capture antibody to an epitope does not interfere withbinding of the detection antibody to an epitope. Either monoclonal orpolyclonal antibodies may be used as the capture and detectionantibodies in the sandwich immunoassay.

Generally, at least two antibodies are employed to separate and quantifyanalyte of interest in a test sample. More specifically, the at leasttwo antibodies bind to certain epitopes of analyte of interest or ananalyte of interest fragment forming an immune complex which is referredto as a “sandwich”. One or more antibodies can be used to capture theanalyte of interest in the test sample (these antibodies are frequentlyreferred to as a “capture” antibody or “capture” antibodies), and one ormore antibodies with a detectable label (e.g., tag attached by acleavable linker) that also bind the analyte of interest (theseantibodies are frequently referred to as the “detection” antibody or“detection” antibodies) can be used to complete the sandwich. In someembodiments, an aptamer may be used as the second binding member and mayserve as the detectable tag. In a sandwich assay, the binding of anantibody to its epitope desirably is not diminished by the binding ofany other antibody in the assay to its respective epitope. In otherwords, antibodies are selected so that the one or more first antibodiesbrought into contact with a test sample suspected of containing analyteof interest do not bind to all or part of an epitope recognized by thesecond or subsequent antibodies, thereby interfering with the ability ofthe one or more second detection antibodies to bind to the analyte ofinterest.

In a preferred embodiment, a test sample suspected of containing analyteof interest can be contacted with at least one capture antibody (orantibodies) and at least one detection antibodies either simultaneouslyor sequentially. In the sandwich assay format, a test sample suspectedof containing analyte of interest (membrane-associated analyte ofinterest, soluble analyte of interest, fragments of membrane-associatedanalyte of interest, fragments of soluble analyte of interest, variantsof analyte of interest (membrane-associated or soluble analyte ofinterest) or any combinations thereof) is first brought into contactwith the at least one capture antibody that specifically binds to aparticular epitope under conditions which allow the formation of anantibody-analyte of interest complex. If more than one capture antibodyis used, a multiple capture antibody-analyte of interest complex isformed. In a sandwich assay, the antibodies, preferably, the at leastone capture antibody, are used in molar excess amounts of the maximumamount of analyte of interest or the analyte of interest fragmentexpected in the test sample.

Optionally, prior to contacting the test sample with the at least onefirst capture antibody, the at least one capture antibody can be boundto a solid support which facilitates the separation the antibody-analyteof interest complex from the test sample. Any solid support known in theart can be used, including but not limited to, solid supports made outof polymeric materials in the form of planar substrates or beads, andthe like. The antibody (or antibodies) can be bound to the solid supportby adsorption, by covalent bonding using a chemical coupling agent or byother means known in the art, provided that such binding does notinterfere with the ability of the antibody to bind analyte of interestor analyte of interest fragment. Moreover, if necessary, the solidsupport can be derivatized to allow reactivity with various functionalgroups on the antibody. Such derivatization requires the use of certaincoupling agents such as, but not limited to, maleic anhydride,N-hydroxysuccinimide, azido, alkynyl, and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

After the test sample suspected of containing analyte of interest isbrought into contact with the at least one capture antibody, the testsample is incubated in order to allow for the formation of a captureantibody (or capture antibodies)-analyte of interest complex. Theincubation can be carried out at a pH of from about 4.5 to about 10.0,at a temperature of from about 2° C. to about 45° C., and for a periodfrom at least about one (1) minute to about eighteen (18) hours, fromabout 2-6 minutes, or from about 3-4 minutes.

After formation of the capture antibody (antibodies)-analyte of interestcomplex, the complex is then contacted with at least one detectionantibody (under conditions which allow for the formation of a captureantibody (antibodies)-analyte of interest-detection antibody(antibodies) complex). If the capture antibody-analyte of interestcomplex is contacted with more than one detection antibody, then acapture antibody (antibodies)-analyte of interest-detection antibody(antibodies) detection complex is formed. As with the capture antibody,when the at least one detection (and subsequent) antibody is broughtinto contact with the capture antibody-analyte of interest complex, aperiod of incubation under conditions similar to those described aboveis required for the formation of the capture antibody(antibodies)-analyte of interest-detection antibody (antibodies)complex. Preferably, at least one detection antibody contains adetectable label (e.g., tag attached by a cleavable linker). Thedetectable label can be bound to the at least one detection antibodyprior to, simultaneously with or after the formation of the captureantibody (antibodies)-analyte of interest-detection antibody(antibodies) complex. Any detectable label known in the art can be used,e.g., a cleavable linker as discussed herein, and others known in theart.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for assay is not critical. If the firstspecific binding partner is detectably labeled (e.g., tag attached witha cleavable linker), then detectably-labeled first specific bindingpartner-analyte of interest complexes form. Alternatively, if a secondspecific binding partner is used and the second specific binding partneris detectably labeled (e.g., tag attached with a cleavable linker), thendetectably-labeled complexes of first specific binding partner-analyteof interest-second specific binding partner form. Any unbound specificbinding partner, whether labeled or unlabeled, can be removed from themixture using any technique known in the art, such as washing.

Next, signal, indicative of the presence of analyte of interest or afragment thereof is generated. Based on the parameters of the signalgenerated, the amount of analyte of interest in the sample can bequantified. Optionally, a standard curve can be generated using serialdilutions or solutions of known concentrations of analyte of interest bymass spectroscopy, gravimetric methods, and other techniques known inthe art.

c) Forward Competitive Inhibition

In a forward competitive format, an aliquot of labeled analyte ofinterest (e.g., analyte having tag attached with a cleavable linker) ofa known concentration is used to compete with analyte of interest in atest sample for binding to analyte of interest antibody.

In a forward competition assay, an immobilized specific binding partner(such as an antibody) can either be sequentially or simultaneouslycontacted with the test sample and a labeled analyte of interest,analyte of interest fragment or analyte of interest variant thereof. Theanalyte of interest peptide, analyte of interest fragment or analyte ofinterest variant can be labeled with any detectable label, including adetectable label comprised of tag attached with a cleavable linker. Inthis assay, the antibody can be immobilized on to a solid support.Alternatively, the antibody can be coupled to an antibody, such as anantispecies antibody, that has been immobilized on a solid support, suchas a microparticle or planar substrate.

Provided herein are methods for measuring or detecting an analytepresent in a biological sample. The method includes contacting thesample with a binding member, wherein the binding member is immobilizedon a solid support and wherein the binding member specifically binds tothe analyte; contacting the sample, which may contain analyte bound tothe binding member, with a labeled analyte, wherein the labeled analyteis labeled with a cleavable tag; removing labeled analyte not bound tothe binding member; cleaving the tag attached to the labeled analytebound to the binding member; translocating the cleaved tag through oracross one or more nanopores in a layer; and assessing the tagtranslocating through the layer, wherein measuring the number of tagstranslocating through the layer measures the amount of analyte presentin the sample, or detecting tags translocating through the layer detectsthat the analyte is present in the sample. In some embodiments,measuring the tags translocating through the layer is assessed, whereinthe number of tags translocating through the layer measures the amountof analyte present in the sample. In some embodiments, detecting thetags translocating through the layer is assessed, wherein detecting tagstranslocating through the layer detects that the analyte is present inthe sample.

Provided herein are methods for measuring or detecting an analytepresent in a biological sample. The method includes contacting thesample with a binding member, wherein binding member is immobilized on asolid support and wherein binding member specifically binds to theanalyte; contacting the sample, which may contain analyte bound to thebinding member, with a labeled analyte, wherein the labeled analytecomprises an aptamer; removing labeled analyte not bound to the bindingmember; dissociating the aptamer bound to the labeled analyte bound tothe binding member and translocating the dissociated aptamer through oracross one or more nanopores in a layer; and assessing the aptamertranslocating through the layer, wherein measuring the number ofaptamers translocating through the layer measures the amount of analytepresent in the sample, or detecting aptamers translocating through thelayer detects that the analyte is present in the sample. In someembodiments, measuring the aptamers translocating through the layer isassessed, wherein the number of aptamers translocating through the layermeasures the amount of analyte present in the sample. In someembodiments, detecting the aptamers translocating through the layer isassessed, wherein detecting tags translocating through the layer detectsthat the analyte is present in the sample.

The labeled analyte of interest, the test sample and the antibody areincubated under conditions similar to those described above inconnection with the sandwich assay format. Two different species ofantibody-analyte of interest complexes may then be generated.Specifically, one of the antibody-analyte of interest complexesgenerated contains a detectable label (e.g., tag) while the otherantibody-analyte of interest complex does not contain a detectablelabel. The antibody-analyte of interest complex can be, but does nothave to be, separated from the remainder of the test sample prior toquantification of the detectable label. Regardless of whether theantibody-analyte of interest complex is separated from the remainder ofthe test sample, the amount of detectable label in the antibody-analyteof interest complex is then quantified. The concentration of analyte ofinterest (such as membrane-associated analyte of interest, solubleanalyte of interest, fragments of soluble analyte of interest, variantsof analyte of interest (membrane-associated or soluble analyte ofinterest) or any combinations thereof) in the test sample can then bedetermined, e.g., as described above. If helpful, determination can bedone by comparing the quantity of detectable label in theantibody-analyte of interest complex to a standard curve. The standardcurve can be generated using serial dilutions of analyte of interest(such as membrane-associated analyte of interest, soluble analyte ofinterest, fragments of soluble analyte of interest, variants of analyteof interest (membrane-associated or soluble analyte of interest) or anycombinations thereof) of known concentration, where concentration isdetermined by mass spectroscopy, gravimetrically and by other techniquesknown in the art.

Optionally, the antibody-analyte of interest complex can be separatedfrom the test sample by binding the antibody to a solid support, such asthe solid supports discussed above in connection with the sandwich assayformat, and then removing the remainder of the test sample from contactwith the solid support.

d) Reverse Competition Assay

In a reverse competition assay, an immobilized analyte of interest caneither be sequentially or simultaneously contacted with a test sampleand at least one labeled antibody.

Provided herein are methods for measuring or detecting an analytepresent in a biological sample. The method includes contacting thesample with a binding member, wherein the binding member specificallybinds to the analyte, and the binding member is labeled with a cleavabletag; contacting the sample, which may contain analyte bound to thebinding member, with a immobilized analyte, wherein the immobilizedanalyte is immobilized on a solid support; removing binding member notbound to the immobilized analyte; cleaving the tag attached to thebinding member bound to the immobilized analyte; translocating thecleaved tag through or across one or more nanopores in a layer; andassessing the tag translocating through the layer, wherein measuring thenumber of tags translocating through the layer measures the amount ofanalyte present in the sample, or detecting tags translocating throughthe layer detects that the analyte is present in the sample. In someembodiments, measuring the tags translocating through the layer isassessed, wherein the number of tags translocating through the layermeasures the amount of analyte present in the sample. In someembodiments, detecting the tags translocating through the layer isassessed, wherein detecting tags translocating through the layer detectsthat the analyte is present in the sample.

Provided herein are methods for measuring or detecting an analytepresent in a biological sample. The method includes contacting thesample with a binding member, wherein the binding member specificallybinds to the analyte, and the binding member comprises an aptamer;contacting the sample, which may contain analyte bound to the bindingmember, with a immobilized analyte, wherein the immobilized analyte isimmobilized on a solid support; removing binding member not bound to theimmobilized analyte; dissociating the aptamer bound to the bindingmember that is bound to the immobilized analyte and translocating thedissociated aptamer through or across one or more nanopores in a layer;and assessing the aptamer translocating through the layer, whereinmeasuring the number of aptamers translocating through the layermeasures the amount of analyte present in the sample, or detectingaptamers translocating through the layer detects that the analyte ispresent in the sample. In some embodiments, measuring the aptamerstranslocating through the layer is assessed, wherein the number ofaptamers translocating through the layer measures the amount of analytepresent in the sample. In some embodiments, detecting the aptamerstranslocating through the layer is assessed, wherein detecting tagstranslocating through the layer detects that the analyte is present inthe sample.

The analyte of interest can be bound to a solid support, such as thesolid supports discussed above in connection with the sandwich assayformat.

The immobilized analyte of interest, test sample and at least onelabeled antibody are incubated under conditions similar to thosedescribed above in connection with the sandwich assay format. Twodifferent species analyte of interest-antibody complexes are thengenerated. Specifically, one of the analyte of interest-antibodycomplexes generated is immobilized and contains a detectable label(e.g., tag attached with a cleavable linker) while the other analyte ofinterest-antibody complex is not immobilized and contains a detectablelabel. The non-immobilized analyte of interest-antibody complex and theremainder of the test sample are removed from the presence of theimmobilized analyte of interest-antibody complex through techniquesknown in the art, such as washing. Once the non-immobilized analyte ofinterest antibody complex is removed, the amount of detectable label inthe immobilized analyte of interest-antibody complex is then quantifiedfollowing cleavage of the tag. The concentration of analyte of interestin the test sample can then be determined by comparing the quantity ofdetectable label as described above. If helpful, this can be done withuse of a standard curve. The standard curve can be generated usingserial dilutions of analyte of interest or analyte of interest fragmentof known concentration, where concentration is determined by massspectroscopy, gravimetrically and by other techniques known in the art.

e) One-Step Immunoassay or Capture on the Fly Assay

In a one-step immunoassay or Capture on the fly assay, a solid substrateis pre-coated with an immobilization agent. The capture agent, theanalyte and the detection agent are added to the solid substratetogether, followed by a wash step prior to detection. The capture agentcan bind the analyte and comprises a ligand for an immobilization agent.The capture agent and the detection agents may be antibodies or anyother moiety capable of capture or detection as described herein orknown in the art. The ligand may comprise a peptide tag and animmobilization agent may comprise an anti-peptide tag antibody.Alternately, the ligand and the immobilization agent may be any pair ofagents capable of binding together so as to be employed for a capture onthe fly assay (e.g., specific binding pair, and others such as are knownin the art). More than one analyte may be measured. In some embodiments,the solid substrate may be coated with an antigen and the analyte to beanalyzed is an antibody.

In some embodiments, a solid support (such as a microparticle)pre-coated with an immobilization agent (such as biotin, streptavidin,etc.) and at least a first specific binding member and a second specificbinding member (which function as capture and detection reagents,respectively) are used. The first specific binding member comprises aligand for the immobilization agent (for example, if the immobilizationagent on the solid support is streptavidin, the ligand on the firstspecific binding member may be biotin) and also binds to the analyte ofinterest. The second specific binding member comprises a detectablelabel and binds to an analyte of interest. The solid support and thefirst and second specific binding members may be added to a test sample(either sequentially or simultaneously). The ligand on the firstspecific binding member binds to the immobilization agent on the solidsupport to form a solid support/first specific binding member complex.Any analyte of interest present in the sample binds to the solidsupport/first specific binding member complex to form a solidsupport/first specific binding member/analyte complex. The secondspecific binding member binds to the solid support/first specificbinding member/analyte complex and the detectable label is detected. Anoptional wash step may be employed before the detection. In certainembodiments, in a one-step assay more than one analyte may be measured.In certain other embodiments, more than two specific binding members canbe employed. In certain other embodiments, multiple detectable labelscan be added. In certain other embodiments, multiple analytes ofinterest can be detected.

The use of a one step immunoassay or capture on the fly assay can bedone in a variety of formats as described herein, and known in the art.For example the format can be a sandwich assay such as described above,but alternately can be a competition assay, can employ a single specificbinding member, or use other variations such as are known.

f) Combination Assays (Co-Coating of Microparticles with Ag/Ab)

In a combination assay, a solid substrate, such as a microparticle isco-coated with an antigen and an antibody to capture an antibody and anantigen from a sample, respectively. The solid support may be co-coatedwith two or more different antigens to capture two or more differentantibodies from a sample. The solid support may be co-coated with two ormore different antibodies to capture two or more different antigens froma sample.

Additionally, the methods described herein may use blocking agents toprevent either specific or non-specific binding reactions (e.g., HAMAconcern) among assay compounds. Once the agent (and optionally, anycontrols) is immobilized on the support, the remaining binding sites ofthe agent may be blocked on the support. Any suitable blocking reagentknown to those of ordinary skill in the art may be used. For example,bovine serum albumin (“BSA”), phosphate buffered saline (“PBS”)solutions of casein in PBS, Tween 20™ (Sigma Chemical Company, St.Louis, Mo.), or other suitable surfactant, as well as other blockingreagents, may be employed.

As is apparent from the present disclosure, the methods and devicesdisclosed herein, including variations, may be used for diagnosing adisease, disorder or condition in a subject suspected of having thedisease, disorder, or condition. For example, the sample analysis may beuseful for detecting a disease marker, such as, a cancer marker, amarker for a cardiac condition, a toxin, a pathogen, such as, a virus, abacteria, or a portion thereof. The methods and devices also may be usedfor measuring analyte present in a biological sample. The methods anddevices also may be used in blood screening assays to detect a targetanalyte. The blood screening assays may be used to screen a bloodsupply.

6. COUNTING AND DATA ANALYSIS

The number of translocation events can be determined qualitatively orquantitatively using any routine techniques known in the art. In someembodiments, the number of translocation events can be determined byfirst calculating the anticipated current change found in a doublestranded DNA translocation event under experimental test conditionsusing the equation:

$\begin{matrix}{{{\Delta G} = \frac{\sigma\pi d_{DNA}^{2}}{4L}},} & ({S1})\end{matrix}$

as referenced in Kwok et al., “Nanopore Fabrication by controlledDielectric Breakdown” Supplementary Information Section 8 and Kwok, H.;Briggs, K.; and Tabard-Cossa, V. “Nanopore Fabrication by ControlledDielectric Breakdown”—PLoS ONE 9(3): e92880 (2014). Using thisanticipated current blockage value, the binary file data of theexperimental nanopore output can be visually or manually scanned foracceptable anticipated current blockage events. Using these events, theThreshold and Hysteresis parameters required for the CUSUM nanoporesoftware can be applied and executed. The output from this software canbe further analyzed using the cusumtools readevents.py software andfiltering blockage events greater than 1000 pA (as determined from thefirst calculation). The flux events, time between events and othercalculations can be determined from the readevents.py analysis tool.Additional calculations can be made on the CUSUM generated data usingJMP software (SAS Institute, Cary, N.C.). Other methods of thresholdsettings for data analysis known in the art can be used.

7. QUALITATIVE ANALYSIS

A qualitative assay can be conducted using the methods and process ofsteps as described herein. A direct assay can be conducted using thecleavable linker conjugate, as described in Example 17, with a thiolbased cleavage step, as shown in FIG. 25. It is understood that othercleavable linker approaches to conducting such an assay may alsoinclude, but are not limited to, various other methods of cleavage of alinker so as to allow for the counting of various tags, as describedherein. Additionally, aptamers can be employed. For example, such otheralternative cleavage methods and/or reagents in addition to the methoddescribed in Example 17 can include those described in Example 16,Example 18, Example 19, Example 20 and Example 21, in addition to othercleavage methods described herein and known to those skilled in the art.It is also understood that while the assay format demonstrated in thisExample (Example 24) represents a direct assay, other formats such assandwich immunoassay formats and/or various competitive assay formats,and including capture on the fly formats, such as are known to thoseskilled in the art, can be implemented as well to conduct an assay usingthe described methods.

For example, the sandwich immunoassay format for the detection of TSH(thyroid stimulating hormone), as described in Example 9, demonstratedthe ability to conduct such an assay on a low cost DMF chip.Additionally, a number of various bioconjugation reagents useful for thegeneration of immunoconjugate or other active specific binding membershaving cleavable linkers can be synthesized using variousheterobifunctional cleavable linkers such as those described in Example1, Example 2, Example 3, Example 4, Example 5 and Example 6, in additionto other cleavable linkers that are otherwise known to those skilled inthe art. Immunoconjugates useful for the practice of the presentinvention can be synthesized by methods such as those described inExample 3, Example 4, Example 5 and Example 6 as well as by othermethods known to those skilled in the art. Additionally, Example 8 showsthe functionality of various fluidic droplet manipulations on a low costchip that can facilitate various steps needed to carry out various assayformats including sandwich and competitive assay formats, and includingcapture on the fly formats, as well as other variations thereof known tothose skilled in the art. Example 11 shows the fabrication of a nanoporethat can be used to count cleavable label in an assay but it isunderstood that other methods for nanopore fabrication known to thoseskilled in the art can also be used for this purpose. Example 16 alsorepresents another construct useful for the conduct of an assay where acleavage is effected, thus leading to a countable label being releasedso as to be countable using the nanopore counting method, as describedwithin this example. This construct and others that would be apparent tothose skilled in the art can be used in an assay as described herein.

Example 22 shows generally how counting can be done so as to be able tomeasure translocation events relating to the presence of a variety oflabels traversing the nanopore. FIG. 29 shows the concept ofthresholding of the signal so as to be able to manipulate the quality ofdata in a counting assay. FIG. 28 shows qualitative assay data that isrepresentative of the type of data that can be used to determine thepresence of an analyte using such assay methods as described within thisexample. It is also understood that while dsDNA was used as a label inthis particular example, other labels, such as the label described inExample 5 and/or Example 22 can also be utilized, including, but notlimited to nanobeads, dendrimers and the like. Moreover, other knownlabels also can be employed. Such constructs as needed to generateappropriate reagents can be synthesized through various examplesdescribed herein in this application, or otherwise via methods known tothose skilled in the art.

8. QUANTITATIVE ANALYSIS

A quantitative assay can be conducted using the methods and process ofsteps as described herein. A direct assay can be conducted using thecleavable linker conjugate, as described in Example 17, with a thiolbased cleavage step, and as shown in FIG. 25. It is understood thatother cleavable linker approaches to conducting such an assay may alsoinclude, but are not limited to, various other methods of cleavage of alinker so as to allow for counting of various tags using a nanopore, asdescribed herein. Additionally, aptamers can be employed. For example,such other cleavage methods in addition to the method described inExample 17 can include, but is not limited to, those described inExample 18, Example 19, Example 20 and Example 21, in addition to othermethods described herein and known to those skilled in the art. It isalso understood that while the assay format demonstrated in this Example(Example 25) represents a direct assay, other formats such as sandwichimmunoassay formats and/or various competitive assay formats, andincluding capture on the fly formats, such as are known to those skilledin the art, can be implemented as well to conduct an assay.

For example, the sandwich immunoassay format for the detection of TSH(thyroid stimulating hormone), as described in Example 9, demonstratedthe ability to conduct such an assay on a low cost DMF chip.Additionally, a number of various bioconjugation reagents useful for thegeneration of immunoconjugate or other active specific binding membershaving cleavable linkers can be synthesized by those skilled in the artusing various heterobifunctional cleavable linkers and conjugatessynthesized by methods such as those described in Example 1, Example 2,Example 3, Example 4, Example 5 and Example 6, in addition to othercleavable linkers or conjugates that could be synthesized by methodsthat are known to those skilled in the art. Additionally, Example 8shows the functionality of various fluidic droplet manipulations on alow cost chip that can facilitate various steps needed to carry outvarious assay formats including sandwich and competitive assay formats,and including capture on the fly formats, as well as other variationsthereof known to those skilled in the art. Example 16 also representsanother construct useful for the conduct of an assay where a cleavage iseffected, thus leading to a countable label being released so as to becountable using the nanopore counting method as described within thisexample. This construct as well as other that would be apparent to thoseskilled in the art can be used in an assay as described herein.

Example 22 shows generally how counting can be performed so as to beable to measure translocation events relating to the presence of a labeltraversing the nanopore. FIG. 29 shows the concept of thresholding ofthe signal so as to be able to manipulate the quality of data in acounting assay. FIGS. 31, 32 and 33 show quantitative assay data outputthat is representative of the type of data that can be used to determinethe amount of an analyte using such assay methods as described withinthis example. FIG. 34 shows a standard curve generated from a constructthat has been cleaved using a chemical method. It is also understoodthat while dsDNA was used as a label in this particular example, otherlabels, such as the label described in Example 5, can also be utilized,including, but not limited to, nanobeads, dendrimers and the like.Moreover, other known labels also can be employed. Such constructs asneeded to generate appropriate reagents can be synthesized as describedherein, or via methods known to those skilled in the art.

9. KITS AND CARTRIDGES

Also provided herein is a kit for use in performing the above-describedmethods with or without the disclosed device. The kit may includeinstructions for analyzing the analyte with the disclosed device.Instructions included in the kit may be affixed to packaging material ormay be included as a package insert. The instructions may be written orprinted materials, but are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this disclosure. Such media include, but are not limitedto, electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. As used herein,“instructions” may include the address of an internet site that providesthe instructions.

The kit may include a cartridge that includes a microfluidics modulewith a built-in nanopore module, as described above. In someembodiments, the microfluidics and nanopore modules may be separatecomponents for reversible integration together or may be fully orirreversibly integrated in a cartridge. The cartridge may be disposable.The cartridge may include one or more reagents useful for practicing themethods disclosed above. The cartridge may include one or morecontainers holding the reagents, as one or more separate compositions,or, optionally, as admixture where the compatibility of the reagentswill allow. The cartridge may also include other material(s) that may bedesirable from a user standpoint, such as buffer(s), a diluent(s), astandard(s) (e.g., calibrators and controls), and/or any other materialuseful in sample processing, washing, or conducting any other step ofthe assay. The cartridge may include one or more of the specific bindingmembers described above.

Alternatively or additionally, the kit may comprise a calibrator orcontrol, e.g., purified, and optionally lyophilized analyte of interestor in liquid, gel or other forms on the cartridge or separately, and/orat least one container (e.g., tube, microtiter plates or strips) for usewith the device and methods described above, and/or a buffer, such as anassay buffer or a wash buffer, either one of which can be provided as aconcentrated solution. In some embodiments, the kit comprises allcomponents, i.e., reagents, standards, buffers, diluents, etc., whichare necessary to perform the assay. The instructions also can includeinstructions for generating a standard curve.

The kit may further comprise reference standards for quantifying theanalyte of interest. The reference standards may be employed toestablish standard curves for interpolation and/or extrapolation of theanalyte of interest concentrations. The kit may include referencestandards that vary in terms of concentration level. For example, thekit may include one or more reference standards with either a highconcentration level, a medium concentration level, or a lowconcentration level. In terms of ranges of concentrations for thereference standard, this can be optimized per the assay. Exemplaryconcentration ranges for the reference standards include but are notlimited to, for example: about 10 fg/mL, about 20 fg/mL, about 50 fg/mL,about 75 fg/mL, about 100 fg/mL, about 150 fg/mL, about 200 fg/mL, about250 fg/mL, about 500 fg/mL, about 750 fg/mL, about 1000 fg/mL, about 10pg/mL, about 20 pg/mL, about 50 pg/mL, about 75 pg/mL, about 100 pg/mL,about 150 pg/mL, about 200 pg/mL, about 250 pg/mL, about 500 pg/mL,about 750 pg/mL, about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about12.5 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 40ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL,about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 165ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250ng/mL, about 275 ng/mL, about 300 ng/mL, about 400 ng/mL, about 425ng/mL, about 450 ng/mL, about 465 ng/mL, about 475 ng/mL, about 500ng/mL, about 525 ng/mL, about 550 ng/mL, about 575 ng/mL, about 600ng/mL, about 700 ng/mL, about 725 ng/mL, about 750 ng/mL, about 765ng/mL, about 775 ng/mL, about 800 ng/mL, about 825 ng/mL, about 850ng/mL, about 875 ng/mL, about 900 ng/mL, about 925 ng/mL, about 950ng/mL, about 975 ng/mL, about 1000 ng/mL, about 2 μg/mL, about 3 μg/mL,about 4 μg/mL, about 5 μg/mL, about 6 μg/mL, about 7 μg/mL, about 8μg/mL, about 9 μg/mL, about 10 μg/mL, about 20 μg/mL, about 30 μg/mL,about 40 μg/mL, about 50 μg/mL, about 60 μg/mL, about 70 μg/mL, about 80μg/mL, about 90 μg/mL, about 100 μg/mL, about 200 μg/mL, about 300μg/mL, about 400 μg/mL, about 500 μg/mL, about 600 μg/mL, about 700μg/mL, about 800 μg/mL, about 900 μg/mL, about 1000 μg/mL, about 2000μg/mL, about 3000 μg/mL, about 4000 μg/mL, about 5000 μg/mL, about 6000μg/mL, about 7000 μg/mL, about 8000 μg/mL, about 9000 μg/mL, or about10000 μg/mL.

Any specific binding members, which are provided in the kit mayincorporate a tag or label, such as a fluorophore, enzyme, aptamer,dendrimer, bead, nanoparticle, microparticle, polymer, protein,biotin/avidin label, or the like, or the kit can include reagents forlabeling the specific binding members or reagents for detecting thespecific binding members and/or for labeling the analytes or reagentsfor detecting the analyte. If desired, the kit can contain one or moredifferent tags or labels. The kit may also include components to elicitcleavage, such as a cleavage mediated reagent. For example, a cleavagemediate reagent may include a reducing agent, such as dithiothreitol(DTT) or tris(2-carboxyethyl)phosphine) TCEP. The specific bindingmembers, calibrators, and/or controls can be provided in separatecontainers or pre-dispensed into an appropriate assay format orcartridge. The tag may be detected using the disclosed device.

The kit may include one or more specific binding members, for example,to detect one or more target analytes in the sample in a multiplexingassay. The number of different types of specific binding members in thekit may range widely depending on the intended use of the kit. Thenumber of specific binding members in the kit may range from 1 to about10, or higher. For example, the kit may include 1 to 10 specific bindingmembers, 1 to 9 specific binding members, 1 to 8 specific bindingmembers, 1 to 7 specific binding members, 1 to 6 specific bindingmembers, 1 to 5 specific binding members, 1 to 4 specific bindingmembers, 1 to 3 specific binding members, 1 to 2 specific bindingmembers, 2 to 10 specific binding members, 2 to 9 specific bindingmembers, 2 to 8 specific binding members, 2 to 7 specific bindingmembers, 2 to 6 specific binding members, 2 to 5 specific bindingmembers, 2 to 4 specific binding members, 3 to 10 specific bindingmembers, 3 to 9 specific binding members, 3 to 8 specific bindingmembers, 3 to 7 specific binding members, 3 to 6 specific bindingmembers, 3 to 5 specific binding members, 3 to 4 specific bindingmembers, 4 to 10 specific binding members, 4 to 9 specific bindingmembers, 4 to 8 specific binding members, 4 to 7 specific bindingmembers, 4 to 6 specific binding members, 5 to 10 specific bindingmembers, 5 to 9 specific binding members, 5 to 8 specific bindingmembers, 5 to 7 specific binding members, 5 to 6 specific bindingmembers, 6 to 10 specific binding members, 6 to 9 specific bindingmembers, 6 to 8 specific binding members, 6 to 7 specific bindingmembers, 7 to 10 specific binding members, 7 to 9 specific bindingmembers, 7 to 8 specific binding members, 8 to 10 specific bindingmembers, 8 to 9 specific binding members, or 9 to 10 specific bindingmembers. Each of the one or more specific binding members may bind to adifferent target analyte and each specific binding member may be labeledwith a different tag and/or aptamer. For example, the kit may include afirst specific binding member that binds to a first target analyte, asecond specific binding member that binds to a second target analyte, athird specific binding member that binds to a third target analyte, etc.and the first specific binding member is labeled with a first tag and/oraptamer, the second specific binding member is labeled with a second tagand/or aptamer, the third specific binding member is labeled with athird tag and/or aptamer, etc. In addition to the one or more specificbinding members, the kits may further comprise one or more additionalassay components, such as suitable buffer media, and the like. The kitsmay also include a device for detecting and measuring the tag and/or anaptamer, such as those described supra. Finally, the kits may compriseinstructions for using the specific binding members in methods ofanalyte detection according to the subject invention, where theseinstructions for use may be present on the kit packaging and/or on apackage insert.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the kit reagents, and the standardization of assays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.One or more of the components may be in liquid form.

The various components of the kit optionally are provided in suitablecontainers as necessary. The kit further can include containers forholding or storing a sample (e.g., a container or cartridge for a urine,saliva, plasma, cerebrospinal fluid, or serum sample, or appropriatecontainer for storing, transporting or processing tissue so as to createa tissue aspirate). Where appropriate, the kit optionally also cancontain reaction vessels, mixing vessels, and other components thatfacilitate the preparation of reagents or the test sample. The kit canalso include one or more sample collection/acquisition instruments forassisting with obtaining a test sample, such as various bloodcollection/transfer devices such as microsampling devices,micro-needles, or other minimally invasive pain-free blood collectionmethods; blood collection tube(s); lancets; capillary blood collectiontubes; other single fingertip-prick blood collection methods; buccalswabs, nasal/throat swabs; 16-gauge or other size needle, circular bladefor punch biopsy (e.g., 1-8 mm, or other appropriate size), surgicalknife or laser (e.g., particularly hand-held), syringes, sterilecontainer, or cannula, for obtaining, storing or aspirating tissuesamples; or the like. The kit can include one or more instruments forassisting with joint aspiration, cone biopsies, punch biopsies,fine-needle aspiration biopsies, image-guided percutaneous needleaspiration biopsy, bronchoaveolar lavage, endoscopic biopsies, andlaproscopic biopsies.

If the tag or detectable label is or includes at least one acridiniumcompound, the kit can comprise at least one acridinium-9-carboxamide, atleast one acridinium-9-carboxylate aryl ester, or any combinationthereof. If the tag or detectable label is or includes at least oneacridinium compound, the kit also can comprise a source of hydrogenperoxide, such as a buffer, solution, and/or at least one basicsolution. If desired, the kit can contain a solid phase, such as amagnetic particle, bead, membrane, scaffolding molecule, film, filterpaper, disc, or chip.

If desired, the kit can further comprise one or more components, aloneor in further combination with instructions, for assaying the testsample for another analyte, which can be a biomarker, such as abiomarker of a disease state or disorder, such as infectious disease,cardiac disease, metabolic disease, thyroid disease, etc.

The present invention has multiple aspects, illustrated by thenon-limiting examples provided herein.

Integrated DMF-Electrochemical/Electrical/Optical Detection Chip,Device, and System

As noted in the foregoing sections, an analyte detection deviceconfigured to operate an analyte detection chip to prepare a sample andto detect an analyte related signal from the prepared sample in theanalyte detection chip is disclosed. The analyte detection chip mayinclude a digital microfluidics (DMF) region and an analyte detectionregion which may overlap or may be spatially separated from the DMFregion. As described in preceding sections, the DMF region and nanoporeregions may be separated into individual devices that are operablyconnected or may be integrated into a single cartridge. Also providedherein is an instrument that operates on the DMF regions and nanoporeregions of a partially or fully integrated device to affect movement ofdroplets and for detecting an electrical signal from the nanopore. Thisinstrument is described in detail in the preceding section. Thisinstrument may further include components for operating a different typeof analyte detection device which device may be a cartridge forconducting clinical chemistry.

In certain cases, clinical chemistry may involve detection ofelectrochemical species or chromogenic reaction product generated byaction of an enzyme on a substrate. For example, the substrate may be ananalyte present in a sample and the enzyme may be specific for theanalyte and may catalytically react with the analyte to generate anelectrochemical species or a colored reaction product. In other cases,clinical chemistry may involve capturing the analyte using a firstbinding member to generate a first complex comprising the analyte andthe first binding member; contacting the complex with a second bindingmember, that binds to the analyte, to generate a second complexcomprising the analyte, the first binding member, and the second bindingmember. The second binding member is conjugated to an enzyme thatgenerates an electrochemical species or chromogenic reaction productupon exposure to a suitable substrate.

In certain embodiments, the analyte detection region of a cartridge forconducting clinical chemistry may include electrodes for detection of anelectrochemical species generated when the analyte is present in thesample. Such cartridges are also referred herein as DMF-electrochemicalchip. In other embodiments, the analyte detection region of a cartridgefor conducting clinical chemistry may be configured for detection of alight signal generated when the analyte is present in the sample. Suchcartridges are also referred herein as DMF-optical chip. It is notedthat a cartridge for conducting clinical chemistry may be referred to asDMF-electrochemical chip.

In yet other embodiments, the cartridge may be a multifunctionalconfigured for electrochemical detection and detection using a nanoporelayer. As such, the instruments disclosed herein can operate on aplurality of single function cartridges as well as on a multifunctioncartridge(s).

The DMF region may be used to transfer a droplet for analysis to adetection region where the droplet will be analysed electrically (e.g.,using a nanopore), electrochemically (e.g., using clinical chemistry),and/or optically (e.g., using clinical chemistry). Optical detection maybe colorimetric detection, turbidometric detection, fluorescentdetection, and/or image analysis. Image analysis may include a detectionof an optical signal from the analyte detection cartridge. Opticalsignal may be a light signal, such as a colorimetric, turbidometric, orfluorescent signal. Electrochemical detection may involve amperometry,coulometry, potentiometry, voltametery, impedance, or a combinationthereof.

The phrases “analyte detection chip,” “analyte detection cartridge,” andthe terms “chip” and “cartridge” are used interchangeably herein torefer to a disposable or reuseable sample processing device compatiblewith the analyte detection instruments disclosed herein. The analytedetection instrument disclosed herein is also referred to as analytedetection device that is used to process a sample in the chips providedhere. In certain embodiments, the analyte detection chip may include afirst substrate and a second substrate, where the second substrate ispositioned over the first substrate and separated from the firstsubstrate by a gap. The first or the second substrate may include aplurality of DMF electrodes. The plurality of DMF electrodes may be anarray or a series of electrodes that are individually controllable foractivation and deactivation. The plurality of DMF electrodes may beoverlayed with an insulating material to electrically isolate the DMFelectrodes. In certain embodiments, the space/gap between the first andsecond substrates may be filled with air or with an inert fluid, such asoil. In certain embodiments, the DMF electrodes may be arranged asdescribed in the preceding sections herein. In exemplary embodiments, aseries of DMF electrodes may be disposed on the first substrate and asingle electrode disposed on the second substrate in a facingconfiguration with the series of electrodes on the first substrate. Theseries of electrodes and the single electrode may be covered with aninsulating layer. In other cases, the series or plurality of electrodeson the first substrate may be configured as co-planar electrodes and thesecond substrate may not include an electrode. Various configurations ofDMF electrodes are described in the preceding sections describing anintegrated microfluidics and nanopore devices. Any of theseconfigurations of DMF electrodes can be present in the additionalcartridges disclosed here.

As described in the preceding sections, the electrodes present in thefirst layer and/or the second layer may be fabricated from asubstantially transparent material, such as indium tin oxide, fluorinedoped tin oxide (FTO), doped zinc oxide, and the like. In addition oneor both substrates may be substantially transparent to facilitateoptical interrogation.

The analyte detection device may contain an optical, electrochemical,and/or electrical means for detecting an optical signal,electrochemical, electrical signal in an analyte detection chip insertedinto the device. In addition the analyte detection device includes meansfor operating the DMF electrodes present in the analyte detection chips.The analyte detection device disclosed herein may include one or aplurality of interfaces for interacting with a cartridge. In certaincases, the cartridge interface may be an insertion area, such as, aslot. In other cases, the interface may be a recess for accepting thecartridge and may be enclosed by a door or lid. The analyte detectiondevice may include a single interface which may be compatible with aplurality of analyte detection chips. For example, an insertion slot maybe compatible with an analyte detection chip that detects anelectrochemical signal, an analyte detection chip that detects anoptical signal, and/or analyte detection chip that detects an electricalsignal. In certain embodiments, the analyte detection device may beconfigured for operating a plurality of analyte detection chipssimultaneously, for example, for detecting the same analyte in differentsamples using multiple chips or for simultaneously detecting multipledifferent analytes in the same sample using multiple different chips. Insuch embodiments, the device may include a plurality of interfaces, suchas, insertion areas.

The analyte detection chips of the present disclosure may optionallyinclude a plasma separation component. In certain embodiments, theplasma separation component may include a filter that captures cellspresent in a whole blood sample, allowing plasma to filter through andbe available for processing into a sample droplet(s) for analysis. Inother embodiments, the plasma separation component may be a fluidicseparation element. Embodiments of analyte detection chips are disclosedbelow. Any of the analyte detection chips described below may optionallyinclude a plasma separation component. In certain cases, the plasmaseparation component may be a commercially available membrane. Incertain embodiments, a commercially available membrane such as thoseavailable from International Point of Care, Inc. (e.g., Primecare™Hydrophilic Asymetric Membranes) or from Pall Corporation (e.g., Vivid™Plasma Separation Membrane) may be used for separating plasma. Incertain cases, the membrane may be integrated into the cartridges of thepresent disclosure. In other embodiments, the chips, instruments, andsystems of the present disclosure may be configured to detect an analytein a whole blood sample.

i. DMF-Electrochemical Detection Cartridge

In certain embodiments, a cartridge disclosed herein includes a DMFregion and an analyte detection region which may overlap or be spatiallysegregated. The DMF region may be used to transfer a droplet foranalysis to a detection region where the droplet will be analysedelectrochemically (for detection of electrochemical species generated ina clinical chemistry assay). Electrochemical analysis is performed byutilizing a working electrode that detects an electrical signalgenerated by an electroactive species generated by the presence of ananalyte in the sample. The detected electrical signal may be quantitatedto determine the presence or concentration of the analyte in the sampleas the electrical signal is proportional to the amount of analytepresent in the sample. Electrochemical detection may involveamperometry, coulometry, potentiometry, voltametery, impedance, or acombination thereof performed by instruments provided in the presentdisclosure.

In certain embodiments, the electrochemical species may be generated byaction of an analyte-specific enzyme on the analyte. In otherembodiments, the electrochemical species may be generated by action ofan enzyme on a substrate. In such embodiments, the enzyme is notspecific to the analyte. Rather, the enzyme is conjugated to a bindingmember that specifically binds to the analyte. In certain embodiments,redox mediators may be included in order to amplify the electricalsignal generated by the electrochemical species. Analyte specificenzymes and redox mediators are well known and may be selected based onthe desired sensitivity and/or specificity.

Electrodes for detection of an electrochemical species may be providedin numerous configurations. Such electrodes may be separate from the DMFelectrodes or may be DMF electrodes that have been modified intoelectrodes for electrochemical sensing. The instruments provided hereininclude electrical circuits that control the electrical power applied tothe array of electrodes as well as the electrodes for electrochemicalsensing. Exemplary configurations of analyte detection chips containingDMF electrodes and electrodes for electrochemical sensing are furtherdescribed below.

FIG. 48A-48F provide a schematic of electrodes present in a chip of thepresent disclosure. FIG. 48A depicts the DMF electrodes 510 that areused to transfer a droplet to a sensor area 511 of the chip. The sensorarea 511 includes a working electrode 512 and a reference electrode 513.FIG. 48B depicts a droplet 514 positioned on the sensor area 511. FIG.48C illustrates the sensor area 511, working electrode 512 and referenceelectrode 513, where the electrodes are semicircular and are disposed ina co-planar configuration. While not shown here, one of the electrodesmay be placed in a facing configuration with the other electrode. Insuch an embodiment, the sensor area for electrochemical detection mayinclude a gap separating the working and reference electrodes where theelectrodes are brought into electrical connection upon translocation ofa droplet into the sensor area. The working and reference electrodes areconnected to contact pads 516 and 517 via leads 515. The contact padsare operably connected to the device that operates the chip. Additionalconfigurations of electrodes for electrochemical detection of an analyteof interest are shown in FIGS. 48D-48E. In FIG. 48D, the workingelectrode 512 is a circular while the reference electrode 513 isarc-shaped and is concentric with the working electrode and encirclesthe working electrode. In FIG. 48E, the sensor area includes threeelectrodes—a working electrode 512, a reference electrode 513, and acounter electrode 518. The droplet and the electrodes are sized suchthat the droplet is in contact with both working and referenceelectrodes (and counter electrode, if present). FIG. 48F depicts therelative sizes of a droplet and a working electrode and that the sizeand shape of the electrode(s) is configured to conform to the dropletsize. It is noted that in this embodiment, the reference electrode ispresent in a facing configuration to the working electrode. The workingelectrode 513 has a first diameter (A) that is smaller than the droplet514 which has a second diameter B. The first diameter A may be about 50μm-1.9 mm. The second diameter B may be about 100 μm-2 mm. Other ratiosof the electrode diameter to the droplet diameter may also be used inthe chips of the present disclosure. In embodiments, where the workingand reference electrodes (and the counter electrode, if present) are ina coplanar configuration, the total area of the electrodes (includingany gaps between the electrodes) may be sized to conform to the dropletdiameter (see FIG. 48B).

In certain embodiments, the electrochemical sensors, such as thosedepicted in FIGS. 48A-48F, may be on a surface opposite the DMF surface,such as on the a single top electrode. In this way, the DMF electrodescan cause a sample droplet to be moved to be in contact with anelectrochemical sensor wherein the sample droplet can be interrogatedwhile also be in contact with a DMF electrode.

FIGS. 49A-49C depict analyte detection chips that include DMF electrodes(510) where a DMF electrode is modified into a sensing electrode (510A,510B, or 510C) suitable for electrochemical detection. In FIG. 49A, aDMF electrode 510A is modified by creating an opening in an insulatinglayer disposed over the DMF electrodes, the opening provides an area forcontact between a droplet and the modified DMF electrode 510A forelectrochemical sensing. In FIG. 49B, a modified DMF electrode 510Bincludes multiple pin-hole openings in the insulating layers coveringthe DMF electrodes for contact between a droplet and the modified DMFelectrode. In FIG. 49C, the DMF electrodes are covered with aninsulating layer that is removable by exposure to light. One or more DMFelectrodes may be exposed to light 511 to remove the insulating layerthereby creating a modified DMF electrode that is not covered by theinsulating layer and can thus contact a droplet. In FIG. 49C, theelectrode 510C is exposed to light to remove the light sensitiveinsulating layer and expose the electrode. A DMF electrode disposed in afacing configuration to the DMF electrodes 510 may also be exposed toprovide a reference electrode. For example, a DMF electrode may bemodified to include an opening or multiple openings at an area in afacing configuration with electrodes 510A, 510B, or 510C.

In another embodiment, the DMF electrodes may be disposed on a firstsubstrate and at least one of the electrodes for electrochemical sensingmay be disposed on a second substrate. In some embodiments, the DMFelectrodes may be disposed on a first substrate and the working andreference electrodes for electrochemical sensing may be disposed on thesecond substrate.

In certain embodiments, the DMF-electrochemical chip may include acapillary region and electrodes for electrochemical sensing may bedisposed in the capillary region. The capillary region may facilitatemovement of a droplet into the capillary region of electrochemicalsensing.

In certain embodiments, the analyte detection chips of the presentdisclosure may include a sensing region as disclosed in U.S. Pat. No.5,200,051. As described in U.S. Pat. No. 5,200,051, a sensing electrodeuseful for determining the presence and/or concentration of analytes ofinterest was provided. The sensing electrode detects electrochemicalspecies generated in response to the analyte by action of an enzyme onthe analyte. Sensing electrode is also referred to as working electrode.As is known in the literature, the generation of the electrochemicalspecies may involve use of a redox mediator. Further, the enzyme and/orredox mediator may be present in a reagent mixture localized at thesensing electrode. In other cases, the enzyme and/or redox mediator maybe introduced into the chip using DMF electrodes to transport a dropletcontaining the enzyme and/or redox mediator from a depot connected tothe chip.

In other embodiments, an immunoassay may be utilized. Briefly, in anexemplary immunoassay, the analyte may be captured by a first bindingmember (e.g., a receptor, an aptamer, or an antibody) that binds to theanalyte. After, an optional wash step, a second antibody that binds tothe analyte may be used to create a complex. An second bonding member(e.g., an antibody or aptamer) may be conjugated to an enzyme, whichenzyme may act on a substrate to generate an electrochemical speciesdetected by the working electrode. In certain cases, the enzyme mayhydrolyze the substrate. This hydrolyzed substrate can then undergoreactions which produce changes in the concentration of electroactivespecies (e.g., dioxygen and hydrogen peroxide) which areelectrochemically detected with the analyte detection chips of thepresent disclosure. Such immunoassays are also exemplified by analkaline phosphatase that is conjugated to a second binding member.Alkaline phosphatase reacts with the substrate(5-bromo-4-chloro-3-indoxyl phosphate) to produce changes in theconcentration of electroactive species (dioxygen and hydrogen peroxide)which are electrochemically detected with the DMF-electrochemicaldetection chip. Both sandwich and competitive assays can be effectedusing the procedures described in U.S. Pat. No. 5,200,051. In theseassays, in addition to the DMF electrode, a working (or sensing)electrode and optional reference electrode may be included. A bioactivelayer may be immobilized on the working electrode, which bioactive layerincludes a first specific binding member (e.g., a receptor or anantibody) that binds to an analyte of interest. In other embodiments,the sample may be processed using the DMF electrodes and transported tothe working/reference electrodes for detection of electrochemicalspecies.

In an embodiment of the present disclosure, the analyte detection chipmay be used to prepare a droplet that includes the electrochemicalspecies. For example, the steps of mixing a sample droplet with adroplet containing an enzyme that acts on the analyte to createelectrochemical species may be conducted by the DMF electrodes of thechip and the droplet (or a portion thereof) containing theelectrochemical species moved to working and reference electrodes fordetection and optionally measurement of the electrochemical species.

In other embodiments, the DMF electrodes may perform the steps of mixinga sample droplet with a droplet containing a first binding member (e.g.,receptor or antibody) conjugated to a magnetic bead. The resultingdroplet may be mixed with another droplet containing a second antibodyconjugated to an enzyme. The resulting droplet may then be mixed with abuffer droplet to wash away any unbound second antibody and the dropletmixed with a droplet containing a substrate for the enzyme and theresulting droplet moved to the working/reference electrode for detectionof electrochemical species generated by the action of the enzyme on thesubstrate. In such embodiments, since the working electrode does notneed to be functionalized by attachment of a binding member (e.g., areceptor, aptamer, or antibody that binds to the analyte), the sameanalyte detection chip can be used for detecting different types ofanalytes by simply loading droplets containing the binding memberspecific for the analyte being detected/measured. Any immunoassay formatsuch as those described in the preceding sections may be used. DMFelectrodes may be utilized for conducting sample preparation forimmunoassay, such as, in the manner described in the preceding sections.

Similar advantages are realized by the disclosed analyte detection chipwhere the analyte is directly detected by action of an enzyme. Forexample, instead of localizing the enzyme (and additional reagents, suchas, redox mediator) on the working/sensing electrode, the dropletcontaining the reagents may be mixed with the sample droplet and theresulting droplet moved to the working/sensing electrode for detectionof the electrochemical species generated by the enzyme when the analyteis present in the sample. As such, the same chip can be used to detectdifferent analytes by simply loading droplets containing the enzyme thatacts on the analyte being detected/measured. For example, enzymes suchas glucose oxidase or dehydrogenase may be used for detection ofglucose; lactate dehydrogenase for detection of lactate; creatinineamidohydrolase, creatinase, or creatine kinase for detection ofcreatine; and the like. In some examples, the glucose dehydrogenase maybe nicotinamide dinucleotide glucose dehydrogenase (NAD-GDH), pyrrolequinoline quinone glucose dehydrogenase (PQQ-GDH) or flavin-adeninedinucleotide glucose dehydrogenase (FAD-GDH). In other examples, theanalyte may be beta-hydroxybutyrate (ketone) and the enzyme may behydroxybutyrate dehydrogenase.

The size and shape of the electrodes required for detection of theelectrochemical species (e.g., working and reference electrodes) can bedetermined empirically or can be based on the literature. For example,the electrodes may be similar to those disclosed in U.S. Pat. No.5,200,051, which is herein incorporated by reference in its entirety.The material of the electrodes may be any material conducive toelectrochemical sensing. Exemplary electrode materials include carbon,platinum, gold, silver, rhodium, iridium, ruthenium, mercury, palladium,and osmium. In certain cases, the working electrode may be a made fromsilver and the reference electrode may be silver/silver halide (e.g.silver chloride).

In certain embodiments, the working electrode (and optional referenceelectrode) may be covered with a selectively permeable layer. Theselectively permeable layer may substantially exclude molecules with amolecular weight of about 120 kDa or more while allowing the freepermeation of molecules with a molecular weight of about 50 kDa or less.

In certain embodiments, interfering electroactive species having amolecular weight above a desired threshold (e.g., above 120 kDa) mayeffectively be excluded from interacting with the working electrodesurface by employing a selectively permeable silane layer described inU.S. Pat. No. 5,200,051. Such a permselective layer, however, allowslower molecular weight electroactive species, like dioxygen and hydrogenperoxide, to undergo a redox reaction with the underlying electrodesurface. Such a perselective layer may be especially useful inamperometric measurement.

In a potentiometric measurement, a polymeric material having functionalgroups and chemical properties conducive to the further incorporation ofcertain ionophoric compounds may be used as a semipermeableion-sensitive film which is established on the working electrode of theanalyte detection chip. The development of a potential at theelectrode-film interface depends on the charge density, established atequilibrium, of some preselected ionic species. The identity of suchionic species is determined by the choice of the ionophore incorporatedin the semipermeable film. An enzyme which is, in turn, immobilized inthe biolayers described herein catalyzes the conversion of a particularanalyte, present in the sample, to the preselected ionic species. Asnoted herein, the enzyme may not be immobilized in the biolayers butrather brought in proximity to the analyte by the DMF electrodestransporting a droplet containing the enzyme to a sample droplet andfusion of the two droplets.

In another aspect, the analyte detection chips of the present disclosuremay include DMF electrodes that are used for transportation and optionalprocessing of a sample droplet and modified DMF electrodes that are usedfor detection of an analytes, such as, ions, e.g., Na²⁺, K⁺, Ca²⁺, andthe like. For detection of ions in a sample, the modified DMF electrodesmay be covered with an ion-selective membrane instead of theion-impermeable insulating layer that covers the DMF electrodes.

Redox Mediators

Representative examples of redox mediators that may be present in a chipof the present disclosure or introduced into a chip of the presentdisclosure via a droplet, include organometallic redox species such asmetallocenes including ferrocene or inorganic redox species such ashexacyanoferrate (III), ruthenium hexamine, etc. Additional suitableelectron transfer agents usable as redox mediators in the sensors of thepresent invention are osmium transition metal complexes with one or moreligands, each ligand having a nitrogen-containing heterocycle such as2,2′-bipyridine, 1,10-phenanthroline, 1-methyl, 2-pyridyl biimidazole,or derivatives thereof. The electron transfer agents may also have oneor more ligands covalently bound in a polymer, each ligand having atleast one nitrogen-containing heterocycle, such as pyridine, imidazole,or derivatives thereof. One example of an electron transfer agentincludes (a) a polymer or copolymer having pyridine or imidazolefunctional groups and (b) osmium cations complexed with two ligands,each ligand containing 2,2′-bipyridine, 1,10-phenanthroline, orderivatives thereof, the two ligands not necessarily being the same.Some derivatives of 2,2′-bipyridine for complexation with the osmiumcation include but are not limited to 4,4′-dimethyl-2,2′-bipyridine andmono-, di-, and polyalkoxy-2,2′-bipyridines, including4,4′-dimethoxy-2,2′-bipyridine. Derivatives of 1,10-phenanthroline forcomplexation with the osmium cation include but are not limited to4,7-dimethyl-1,10-phenanthroline and mono, di-, andpolyalkoxy-1,10-phenanthrolines, such as4,7-dimethoxy-1,10-phenanthroline. Polymers for complexation with theosmium cation include but are not limited to polymers and copolymers ofpoly(l-vinyl imidazole) (referred to as “PVI”) and poly(4-vinylpyridine) (referred to as “PVP”). Suitable copolymer substituents ofpoly(l-vinyl imidazole) include acrylonitrile, acrylamide, andsubstituted or quaternized N-vinyl imidazole, e.g., electron transferagents with osmium complexed to a polymer or copolymer of poly(l-vinylimidazole). Embodiments may employ electron transfer agents having aredox potential ranging from about −200 mV to about +200 mV versus thestandard calomel electrode (SCE).

Enzymes

The enzymes used in conjunction with the analyte detection chips of thepresent disclosure may be selected based upon the analyte being detectedor the substrate being utilized (e.g., in an immunoassay). Non-limitingexamples of analyte-specific enzymes include one or more of glucoseoxidase, glucose dehydrogenase, NADH oxidase, uricase, urease,creatininase, sarcosine oxidase, creatinase, creatine kinase, creatineamidohydrolase, cholesterol esterase, cholesterol oxidase, glycerolkinase, hexokinase, glycerol-3-phosphate oxidase, lactate dehydrogenase,alkaline phosphatase, alanine transaminase, aspartate transaminase,amylase, lipase, esterase, gamma-glutamyl transpeptidase, L-glutamateoxidase, pyruvate oxidase, diaphorase, bilirubin oxidase, and theirmixtures.

ii. DMF-Optical Chips

In certain embodiments, the analyte detection chips may be used togenerate an optical signal indicating presence of an analyte in a samplebeing assayed by the chips. The optical signal may be, for example, acolorimetric signal, turbidometric signal, and/or a fluorescent signal.The magnitude of the optical signal may be proportional to the amount ofanalyte and may be used to determine the presence or concentration ofthe analyte in the sample.

In certain embodiments, at least one of the substrates of the analytedetection chip may be transparent to facilitate detection of opticalsignal. In addition, the DMF electrodes may be transparent.

The DMF electrodes may be used to process a sample droplet forgeneration of an optical signal indicative of presence of the analyte inthe sample. The optical signal may generated by action of an enzyme on asubstrate. Any assay format may be utilized for generation of an opticalsignal, such as, colorimetric assay (e.g., detect a chromogenic reactionproduced in a clinical chemistry assay), immunoassay, sandwichimmunoassay (e.g., monoclonal-polyclonal sandwich immunoassays,including enzyme detection (enzyme immunoassay (EIA) or enzyme-linkedimmunosorbent assay (ELISA), competitive inhibition immunoassay (e.g.,forward and reverse), enzyme multiplied immunoassay technique (EMIT),particle-enhanced turbidimetric inhibition immunoassay (PETINIA),homogeneous enzyme immunoassay (HEIA), a competitive binding assay,bioluminescence resonance energy transfer (BRET), one-step antibodydetection assay, homogeneous assay, heterogeneous assay, capture on thefly assay, etc. Exemplary assay formats are described herein.

Optical signals that may be measured include fluorescence,chemiluminescence, colorimetric, turbidimetric, etc. In certainembodiments, optical signals may be detected using a spectrophotometer.For example, an optical signal may be detected as described in AnalBioanal Chem (2015) 407:7467-7475, which is herein incorporated byreference in its entirety. In this technique, a custom manifold alignsoptical fibres with a digital microfluidic chip, allowing opticalmeasurements to be made in the plane of the device. Because of thegreater width vs. thickness of a droplet on-device, the in-planealignment of this technique allows it to outperform the sensitivity ofvertical absorbance measurements on digital microfluidic (DMF) devices.In other embodiments, the optical signal may be measured at a planeperpendicular to the chip.

In certain embodiments, the DMF-optical cartridge may include a built-inor a separate component for illuminating a droplet in the cartridge. Abuilt-in or a separate component may also be used for detecting lightfrom the illuminated droplet. For example, a waveguide may be used toilluminating a droplet in the cartridge. A waveguide may also be usedfor detecting an optical signal from the droplet. In certain cases, aregion of the DMF-optical cartridge may be manufactured from a waveguidematerial. In certain cases, one or both substrates of the DMF-opticalcartridge may be a waveguide. Any suitable waveguide that can propagatelight with minimal loss may be used in such cartridges.

The optical signal generation may involve illuminating a droplet with alight source and measuring the light from the droplet using a detector,such as, a spectrometer or a CMOS detector.

An exemplary chip for optical detection of a signal generated by actionof an enzyme is illustrated in FIG. 7 of Anal Bioanal Chem (2015)407:7467-7475. FIG. 7 is reproduced herein as FIG. 50. As shown in FIG.50, the DMF chip includes a PPM disc on which an analyte is absorbed.The DMF chip was used to extract the analyte fluorescein from PPM discin the reaction zone and moved to the detection zone and positionedadjacent an optical fiber. Laser was used to excite the fluorescein andits emission was measured using the optical fiber.

In certain embodiments, the DMF-optical chip may not be configured withan optical fiber. In these embodiments, the droplet may be interrogatedfrom a vertical direction and either reflected light, emitted light orabsorbance from the droplet measured.

iii. DMF-Electrical Detection Chip

Also provided herein are DMF chips that are configured for analyteanalysis using a nanopore layer as described in the preceding sections.Such a chip may be processed using an instrument that includes circuitsconfigured for operating the activation and deactivation of theelectrodes for processing the sample and for measuring a signal at ananopore layer, which signal is generated when a tag or ananalyte-specific binding member (e.g., an aptamer) traverses through thenanopore and which signal is indicative of presence of an analyte in thesample and may be proportional to the concentration of the analyte inthe sample.

iv. DMF-Imaging Chip

Also provided herein are DMF chips that are configured for imageanalysis. The DMF chip may include an array of electrodes (individuallyor collectively energizable) on a first substrate which electrodes arecovered with an insulating layer. The first substrate may be spacedapart from a second substrate. In certain cases, the second substratemay include a ground electrode in a facing configuration to the array ofelectrodes.

The two substrates are separated by a gap. In certain cases, thesubstrates are separated by a narrow gap of about 5 μm or less, such as1 μm. In certain cases, a portion of the DMF chip may include a regionwhere the substrates are separated by a narrow gap of about 5 μm orless, such as 1 μm. For example, the gap between the substrates in aregion where the sample is introduced may be relatively wider (about 100μm), while the gap where the sample droplet (or a processed sampledroplet) is to be imaged in narrower. The smaller gap height results increation of a monolayer of particles that can be imaged and analyzed,therefore making analysis of single particles more straightforward.

FIG. 51A shows a possible representation where red blood cells (RBC) arerepresented by the ellipses. The gap height restricts RBCs from formingmultiple layers within the gap. The image sensor located above the DMFchip is used to collect optical data for analysis. In this embodiment,the illumination is co-located with the image sensor. The top substratemust be optically clear for illumination and imaging. Out of plane fromthe DMF chip is an imaging detector that is used to collect optical datafor analysis. The imager technology can include, but not limited to CMOSand CCD technologies.

FIG. 51B depicts a DMF-imaging chip where a portion of the chip includessubstrates separated by a larger gap which is connected to a capillaryflow region having a gap dimensioned to disperse the particles into asingle layer. The DMF portion of the chip is useful to actively controlfluid flow, mix fluids, move or separate particles to different activereagent areas on the chip or other actions that are useful foranalytical operations (dilutions, etc.) whereas the capillary flowregion is a channel that creates the particle monolayer by the flow gaptransition from a droplet present on the DMF portion of the chip to thenarrow capillary gap due to capillary forces. This allows for materialspresent in droplets to be analyzed via an imaging detector that ispositioned and focused on the capillary flow region. The DMF portion ofthe chip controls fluids and the capillary flow region creates ananalytical region for colorimetric, absorbance, transmission,fluorescence particle counting and imaging (such as cells). The countingand imaging in the capillary channel can be done with either a staticposition of the particles or as the particles flow through the channel.

The gap within the DMF region in the chip of FIG. 51B does not need tobe constrained to a gap height for monolayer of particulate matter,rather, since the imaging detector is focused on the capillary flowregion, the gap of the capillary flow region is held at a value thatprevents multiple layers from forming. The imaging detector assembly canhave multiple depths of fields and movement of the lenses to focus onthe contents within the capillary channel or accommodate differentcapillary heights.

FIGS. 52A and 52B depict embodiments of integrated sample preparationand sample analysis cartridges. A schematic of a cartridge 1000 with aDMF element and an imaging chamber is provided in FIG. 52A. A firstsubstrate 1001 including DMF electrodes 1003 is disposed in a spacedapart manner from a second substrate 1002. The space between the firstand second substrates varies such that a first region of the cartridgeincludes a first chamber having a height h₁ and a second region includesa second chamber having a height h₂. As depicted in FIG. 52A, h₁ islarger than h₂. In certain embodiments, h₁ may range from 20-200 μm(microns), e.g., 50-200 microns, 75-200 microns, 100-200 microns,125-200 microns, 100-175 microns, e.g, 150 microns and h₂ may range from2-10 μm (microns), e.g., 2-8 microns, 2-6 microns, 3-6 microns, e.g., 4microns. Aspects of the disclosed cartridge 1000 include embodimentswhere the first chamber is configured for actuating a sample droplet,e.g., a blood droplet to move the blood droplet in the first chambersuch that the droplet contacts reagents disposed in the first chamber,thereby facilitating processing of the sample and preparation forsubsequent analysis in the second chamber. For example, the firstchamber may include reagents for staining of cells present in a bloodsample to facilitate cell detection/counting, complete blood countand/or other hematology measurements, e.g., staining, counting, and/ormorphological analysis of bacteria, RBCs, WBCs, and/or platelets, etc.As disclosed herein, the DMF electrodes may be operated to move thesample droplet to a region in the first chamber having a reagent (e.g.,a staining reagent, such as, a dye that binds to nucleic acid, e.g.,acridine orange, ethidium bromide, TOTO, TO-PRO, or SYTOX) disposed in adry form or in form of a droplet. The sample droplet may be mixed withthe reagent to provide uniform distribution of the reagent in the sampledroplet. Mixing may be performed by splitting and merging the sampledroplet till at least 80% of the staining reagent is uniformlydistributed within the sample droplet. The second chamber may betransparent at least in an imaging region 1004 to facilitate opticalanalysis of a sample transposed into the second chamber from the firstchamber. As shown, the second chamber may be configured to facilitatedistribution of cells present in the sample as a monolayer, avoidingoverlapping cells which tend to introduce error in optical analysis ofthe cells. The second substrate 1002 may be include a first planarregion and a second planar region separated by a sloping region thatintroduces a shoulder or a step element 1006 for changing the height ofthe plane of the first planar region with reference to the second planarregion. The two-tiered second substrate is disposed over the firstsubstrate that is substantially planar to provide the cartridge thatincludes the two chambers of different heights. As discussed herein, thesample may be moved from the first chamber into the second chamber bycapillary action, DMF electrodes, SAW, or other methods.

FIG. 52B provides a schematic of an embodiment of a cartridge 2000comprising a first chamber 2001 defined by a first substrate 2002 and asecond substrate 2003, spaced apart by a spacer having a height h₃. Thecartridge 2000 also includes a second chamber 2004 defined by a thirdsubstrate 2006 and a fourth substrate 2005 spaced apart by beads 2007having a height h₄. The first substrate is depicted with DMF electrodes2008 although the DMF electrodes may be present on the second substrateor on both substrates, as described herein. Similar to the cartridge inFIG. 52A, the first chamber has a height that is larger than that of thesecond chamber. In certain embodiments, h₃ may range from 20-200 μm(microns), e.g., 50-200 microns, 75-200 microns, 100-200 microns,125-200 microns, 100-175 microns, e.g, 150 microns and h₄ may range from2-10 μm (microns), e.g., 2-8 microns, 2-6 microns, 3-6 microns, e.g, 4microns. The cartridge depicted in FIG. 52B includes polystyrene beadsdispersed between the third and fourth substrates for defining a uniformheight in the second chamber for facilitating distribution of cells as amonolayer. At least a portion of the second chamber may be transparentto facilitate interrogation by an optical device 2010. Similar to thecartridge in FIG. 52A, the first chamber actuates the sample 2012 forpreparation for analysis (e.g., by mixing with a staining reagent) inthe second chamber. Cells 2015 dispersed in a monolayer in the secondchamber are also depicted. The optical device may be positioned tointerrogate the sample through the third or the fourth substrate. Incertain embodiments, the first 2002 and third 2006 substrates may beformed from a single substrate such that the cartridge has a commonbottom substrate. The first and second chambers may be configured toallow for a sample to move from the first chamber to the second chamberutilizing capillary action, DMF electrodes, SAW, or other methods.

Additional configurations for the DMF chamber and the imaging chamberinclude embodiments depicted in FIGS. 52C-52E. The cartridge may includea DMF chamber that includes DMF electrodes for sample preparation (e.g.,mixing a sample droplet with a staining reagent) operably connected toan imaging chamber. As noted in the descriptions for FIGS. 52A and 52B,the height (h₁) of the DMF chamber may range from 20-200 μm (microns),e.g., 50-200 microns, 75-200 microns, 100-200 microns, 125-200 microns,100-175 microns, e.g, 150 microns and the height (h₂) of the imagingchamber may range from 2-10 μm (microns), e.g., 2-8 microns, 2-6microns, 3-6 microns, e.g., 4 microns. FIG. 52C depicts a cartridge 1100a comprising a DMF chamber defined by a first substrate 1101 disposedover a second substrate 1102. The DMF electrodes are not illustrated andmay be present on the first and/or second substrate. The secondsubstrate 1102 extends to the imaging chamber which is defined by thesecond substrate 1102 and third substrate 1104. A spacer 1103 definesdistal end of the DMF chamber. The spacer 1103 may contact a lowersurface of the first substrate 1101 and an upper surface of substrate1104. FIG. 52D depicts a cartridge 1100 b in which the imaging chamberis operably connected to the DMF chamber via a two-part spacer 1103a-1103 b, where a first part of the spacer (1103 a) is disposed betweena first substrate 1101 and a third substrate 1104 and a second part ofthe spacer (1103 b) is disposed between a second substrate 1102 and afourth substrate 1105. FIG. 52E depicts a cartridge 1100 c in which theimaging chamber is disposed in the distal region of the DMF chamber. TheDMF chamber is defined by substrates 1101 and 1102. The imaging chamberis defined by the substrate 1101 and substrate 1104. The spacer 1103supports the substrate 1104.

As noted herein, the DMF chamber may be reversibly coupled to theanalyte detection region (electrochemical detection, electricaldetection, optical detection, etc.) to form an integrated or semiintegrated cartridge. The coupling of the DMF chamber and the analytedetection region may be performed as described herein.

In certain embodiments, the cartridge may include reagents for analysisof a blood sample and may be configured as disclosed in U.S. Pat. Nos.6,004,821 or 8,367,012, which are herein incorporated by reference intheir entirety. In certain embodiments, the DMF electrodes and thechamber for sample preparation may be configured as disclosed inWO2016/161400, WO2016/161402, or US2015/0298124, which are hereinincorporated by reference in their entirety. It is understood thatinstead of or in addition to the DMF electrodes, the cartridges may beconfigured for actuating sample droplets by SAW.

v. DMF Chip with Multiple Detection Regions

Also provided herein is a DMF chip that allows for multiple analyses andtechniques to be utilized on a single DMF chip. FIG. 53 shows a DMF chiplayout where specific detection zones have been created, not utilizing asingular detection technology, but rather, creating zones that areconfigured for the detection technology itself. For example, the zones550 a may be created and configured to allow for electrochemicaldetection; the zones 550 b are specific to imaging analysis, and zones550 c are for absorbance based measurements.

The chip depicted in FIG. 53 provides a compact DMF chip on whichmultiple typical diagnostic tests utilizing different detectiontechnologies can be carried out. For example, hematology measurementstypically rely upon imaging analyses, whereas clinical chemistry orimmunoassay measurements typically rely upon electrochemical or opticalbased detections. Using this chip, a user can utilize a single bloodcollection across multiple diagnostic analytic devices, thus greatlysimplifying the diagnostic process and time to result for users.

FIG. 54 illustrates a DMF chip layout comprised of imaging, photon andelectrochemical sensing. The DMF configuration can be any combinationand/or number of sensing zones depending on the diagnostic requirements.Imaging sensors such as CMOS technology may be utilized. Where highersensitivity is required CCD or enhanced CCD (eCCD) can be used. CMOSdetectors are versatile such that they can be used as electrochemicalsensor with the proper coatings. Where high sensitivity photon sensingis required (listed from highest to lowest) Photomultiplier Tubes (PMT)or Avalanche Photodiode Detectors (APD) or photodiodes can be used.Illumination may be achieved by using Light Emitting Diodes (LED) orsolid state type lasers. The illumination configurations shown,addresses both brightfield and fluorescence excitation. The dichroic orbeam splitter reflects the fluorescent excitation wavelengths andtransmits emission wavelengths. Bandpass wavelengths in the dichroicwill allow for transmitted brightfield wavelengths to be transmitted tothe sensor. Dichroic optical component is not needed for transmittedlight analysis, only fluorescence. Additional excitation and emissionfilters may be required for the different assays and can be included inthe respective optical paths. An analyte detection instrument compatiblewith such a chip may be configured to include means for detecting anoptical signal and an electrical signal. For example, the analytedetection instrument may include imaging sensors such as CMOS, CCD orenhanced CCD (eCCD) camera, PMT, APD. In addition, the analyte detectioninstrument may include means for sample illumination such as LED,lasers, and the like.

An embodiment of a DMF chip with multiple detection regions is shown inFIG. 55. The chip in FIG. 55 includes a sample acquisition port throughwhich a 20-80 μl whole blood sample is loaded onto the chip andimmediately transferred to the re-suspension area where the sample canreside up to ½ hour or more. The re-suspension area is used tore-suspend the blood before it is distributed to the 5 aliquots whichare 0.5-1 μl in volume. Re-suspension is achieved by reciprocating theblood sample such that the fluid path allows for total inversion of theblood, fluid path >2× the length of the blood slug.

The re-suspended blood is transferred and divided into several aliquots.Individually, each aliquot is transferred to a sensing/imaging areaspassing through a reagent section to stain, sphere or lyse the cells.Mixing is accomplished by reciprocating motion of the aliquot in thisregion. The diagram illustrates the different reagents and imaging areasfor hematology measurements. All reagents in the consumable are dried.Other coatings in the fluid path provide hydrophobic and hydrophilicsurfaces for maneuvering the liquid. Alternative configurations may beincluded for conducting electrochemical sensing and optical sensing.Platelets (PLT), Reticulocytes (RETC) and Nucleated Red Blood Cells(NRBC) can be imaged in the same imaging areas as Red Blood Cells (RBC)and White Blood Cells (WBC). DMF may be used for collecting the samplein the acquisition port, re-suspending the sample, optionally includethe aliquot partioning, and stain the entire sample. Such chips may beused for assaying blood agglutination, for example, determining bloodtype.

FIGS. 56 and 57 illustrate DMF chips capable of electrochemical andoptical detection. A 20-80 μl whole blood sample is acquired from apatient at the fill port and immediately transferred to there-suspension area where the sample can reside up to ½ hour or longer.The re-suspension area is used to re-suspend the blood before it isdistributed. Re-suspension is achieved by reciprocating the blood samplesuch that the fluid path allows for total inversion of the blood, fluidpath >2× the length of the blood slug. The sample is transferred anddivided into two aliquots; one for plasma separation and the other forwhole blood analysis. Plasma separation can be achieved with aseparation medium; i.e. filtering, or by fluidic methods utilizing thecapabilities of DMF. The consumable utilizes the same imaging sensorlayout as the hematology construct in FIG. 55. Each imaging area has acorresponding area for reagent mixing. All reagents in the “Staining &Mixing Areas” are dried. To provide sample wash, reagent packs for washmay be added to the fluidics design (FIG. 56).

vi. Analyte Detection Device

As noted herein, an analyte detection device that includes a cartridgeinterface for interacting with the analyte detection chips is provided.In certain embodiments, the analyte detection device may be compatiblewith only one type of analyte detection chip. Such an analyte detectiondevice may include a single cartridge interface, e.g, a single insertionslot and may operate on a single chip inserted into the slot. In otherembodiments, the analyte detection device may include a plurality ofcartridge interfaces, e.g., insertion slots that may be used to operatea plurality of chips (e.g., of the same type, loaded with differentsamples). In yet other embodiments, the analyte detection instrument mayinclude a single cartridge interface in which a single analyte detectionchip may be inserted. In some embodiments, the analyte detection devicemay be a multi-functional instrument or a universal instrument that canoperate upon a plurality of different types of analyte detection chips,e.g, two or more of DMF-electrochemical detection chip (e.g. forclinical chemistry); DMF-optical detection chip (e.g., for clinicalchemistry); DMF-electrical chip (e.g., comprising a nanopore layer); andDMF chip with multiple detection regions. In some embodiments, theuniversal instrument may include a separate insertion slot for eachdifferent analyte detection chip. In other embodiments, the universalinstrument may have a single insertion slot that is compatible with thedifferent types of analyte detection chips. A multi-functional oruniversal analyte detection instrument may include optical detectionunit and electrical detection unit.

The analyte detection device may include a power source and circuits foractuating the DMF electrodes. Depending upon the analyte detection chipthat the device operates upon, the analyte detection device may includecircuits for detecting electrical signals from the working electrode(for electrochemical detection); circuits for detecting electricalsignals from a nanopore; optical detection which may include sensors fordetecting light signals and/or camera(s) for imaging; and combinationsthereof. The analyte detection device may include a memory or may beoperably connected to a memory storing instructions for operation of theanalyte detection chips. In certain cases, the devices may be operatedby a processor that runs a program for carrying out the steps requiredfor generating an analyte related signal and detecting the signal. Theanalyte detection device may also include algorithm(s) to calculate aconcentration of the analyte based on the detected electrical or lightsignal.

The analyte detection devices disclosed herein may also be configured tooperate upon DMF-nanopore devices, such as those disclosed inPCT/US2016/025787, which is herein incorporated by reference in itsentirety. In certain embodiments, the DMF part of theDMF-electrochemical detection chips, DMF-optical detection chips and thelike may be configured and formed as disclosed in PCT/US2016/025785 orPCT/US2016/025787.

FIGS. 58A and 58B depict an analyte detection device. Analyte detectiondevice 410 a in FIG. 58A is compatible with a single type of analytedetection chip. The device 410 a in FIG. 58A may include a singleinterface, such as a single slot 411 a for a single type of analytedetection chip. In certain cases, the device 410 a in FIG. 58A mayinclude multiple interfaces, such as slots (411 a, 411 b, 4111 c, 411 d)that each accept the same type of analyte detection chip. The device 410a in FIG. 58A may be used for simultaneously analyzing multiple samplesfor presence of an analyte. In certain embodiments, the device in FIG.58A may include a housing that includes processor 413 which is operablyconnected to a memory that contains programming for using the detectionchips. The slot 411 a and additional slots (if present) may all becontained in the housing. In other embodiments, the housing may onlyinclude the processor and may optionally include a screen or a monitorand hardware and software sufficient for connecting to and operating aseparate device comprising one or more slots, such as, slots 411 a-411d. Thus, in some embodiments, the operating system of the device may bephysically separable from the slots into which cartridges are placed.

The device 410 b in FIG. 58B includes multiple slots, 412 a, 412 b, 412c, and 412 d. Slot 412 a is compatible with a DMF-electrochemicaldetection chip. Slot 412 b is compatible with a DMF-optical detectionchip. Slot 412 c and 412 d are compatible with a DMF-nanopore and aDMF-clinical chemistry chip, respectively. The DMF-clinical chemistrychip may have an electrochemical detection region or an opticaldetection region. The devices 410 a and 410 b also include a processor413 which is operably connected to a memory that contains programmingfor using the detection chips. Similar to the device 410 a, device 410 bmay include a housing containing the processor 413, an optional screenor a monitor and the slots 412 a-412 d or the housing may not includethe slots 412 a-412 d, which may be present in a separate device(s)connected to the device 410 b.

FIG. 58C depicts an analyte detection chip compatible with the analytedetection device shown in FIGS. 58A and 58B. In certain embodiments, thedevices and systems described herein may include a cartridge adaptor (s)that can be utilized to adapt a single slot to different types ofcartridges. For example, a cartridge adapter 1 may include a firstinterface for connecting to a slot 1 and a second interface forconnecting to a cartridge 1, a cartridge adapter 2 may include a firstinterface for connecting to a slot 1 and a second interface forconnecting to a cartridge 2. Cartridge adapters and cartridgescompatible with the cartridge adaptors are depicted in FIGS. 58D and58E. FIG. 58D illustrates a cartridge adapter 5812 a that includes afirst interface comprising pins 5810 a and 5810 b compatible with a slotpresent in a device that either includes a processor or is connectable(physically or wirelessly) to a processor with instructions forperforming the steps required for preparing a sample in the DMF regionof a cartridge and/or analyzing the prepared sample (e.g., detectinganalyte related signal). The second interface of the cartridge adapter5812 a includes a port 5811 that mates with pin 5813 present on acartridge 5814 (e.g., an immunoassay cartridge). FIG. 58E illustrates acartridge adapter 5812 b that is compatible with the same slot that wascompatible with cartridge adapter 5812 a due to presence of pins 5810 aand 5810 b. However, the second interface of the cartridge adapter 5812b includes a cavity that accommodates and is connectable to cartridge5815 (e.g., a hematology cartridge) but not to cartridge 5814. Thus, acartridge adaptor may be used to adapt a slot to connect with multipledifferent types of cartridges.

FIG. 59A depicts an analyte detection chip that can be used forconducting the analysis of a sample according to the methods describedherein. The analyte detection chip includes an opening in a distalregion which opening provides an inlet (marked with an arrow in FIG.59A) for introducing a sample into the analyte detection chip. As notedherein, in certain cases, the sample may be a whole blood sample. Incertain embodiments, the sample may be pipetted into the opening of theanalyte detection chip. In other embodiments, a sample droplet may bedirectly loaded into the analyte detection chip from a lanced area ofthe skin, such as, a finger tip. The distal region of the analytedetection chip may include elements for processing the sample and/ortransferring the sample to appropriate regions in the analyte detectionchip for detection of one or more analytes present in the sample. Theproximal region of the analyte detection chip is insertable into ananalyte detection device for sample analysis. In certain cases, theanalyte detection chip may include a cover, where a part of the cover atthe proximal region of the chip is moveable to expose the interior ofthe proximal region. The movable portion of the cover may be hingedlyattached to the cover of the chip and may be pivoted up to expose theinterior of the chip. In other cases, the movable portion of the covermay be slidable towards the distal region to expose the interior of thechip at the proximal region. In certain cases, the analyte detectionchip may be compatible with the analyte detection device shown in FIG.59B. As noted herein, the chip may include nanopores and/or nanowells.In addition, the chip may include electrodes, e. g., an array ofelectrodes for digital microfluidics.

FIG. 59B depicts an analyte detection device compatible with the analytedetection chips as described herein. For example, the analyte detectiondevice is compatible with the analyte detection chip shown in FIG. 59A.The analyte detection device of FIG. 59B includes a single insertionslot (indicated by an arrow) into which at least a proximal region of ananalyte detection chip is inserted. In certain cases, the entire orsubstantially the entire chip is inserted into the insertion slot. Insome cases, this analyte detection device may also be configured toinclude multiple interfaces, such as multiple insertion slots. Asdepicted in FIG. 59B, the analyte detection device has an ideal size fora benchtop device with the height in the range of about 12 inches.

The chips, devices, and systems disclosed herein provide many advantagesin the field of sample analysis. These chips and devices are highlyreliable even for small sample volumes and are low cost alternatives toother sample analysis devices. In addition to the small footprint of thedevice, these devices are easy to use and can be used to performmultiple core lab tests, including immunoassay and/or clinicalchemistry. As explained herein, the disclosed chips, device, and systemsprovide high sensitivity which enables analysis of small sample volumes.Furthermore, the configuration of the chip and the device requiresminimal user input and enables a minimally trained user to operate thedevice and chip for analyzing a sample. The chips and devices of thepresent disclosure have no or minimal moving parts which also reducemanufacturing and/or maintenance costs and while increasing life of thedevice.

The analyte detection instrument may include imaging sensors such asCMOS, CCD or enhanced CCD (eCCD) camera, PMT, APD. In addition, theanalyte detection instrument may include means for sample illuminationsuch as LED, lasers, and the like. An analyte detection instrument mayalso include electrical circuits for operating a DMF chip and foroperating a DMF-electrochemical/electrical chip.

In some embodiments, analyte detection may require a certain level ofsensitivity. Depending upon the desired sensitivity, a DMF-optical chip(e.g., a DMF-clinical chemistry chip) or a DMF-electrochemical (e.g., aDMF-clinical chemistry chip) or DMF-electrical (e.g., a DMF-nanoporechip) chip may be utilized. In yet other assays for analyte detection aDMF-imaging chip for example, where a droplet present on the DMFelectrodes is optically interrogated (e.g, using a spectrophotometer)may be used.

vii. Analyte Detection Systems

Also disclosed herein are systems that include the analyte detectionchips and analyte detection instrument compatible with the chips. Asnoted in this disclosure, the instrument can perform multiple assaysusing a single multi-functional chip or using different chips. Forexample, the instrument can detect electrical signals (such as thosefrom an electrochemical species in contact with the working andreference electrodes in a DMF-clinical chemistry chip or from atag/analyte-specific binding member traversing a nanopore in aDMF-nanopore chip) and optical signals including imaging DMF-opticalchip, detecting analyte related signals from a DMF-optical chip, and/orimaging a droplet on a DMF-imaging chip. As noted herein, in aDMF-electrochemical chip, the DMF electrodes may be adjacent to theworking and reference electrodes on a single substrate or the workingand reference electrodes may be disposed in a capillary fluidicallyconnected to the DMF electrodes containing region of the chip. In somecases, the location of the array of DMF electrodes on a DMF-electricalchip with reference to the location of nanopore layer may be asdescribed in the foregoing sections.

The systems of the present disclosure may be programmed for performing amenu of tests for analysis of analyte(s) in a sample. For example, theinstrument may detect the type of chip placed in the instrument and mayselect the assay to be performed on the chip. The instrument mayactivate and deactivate the DMF electrodes to process a sampledroplet(s) and generate a droplet that can be interrogated electricallyor optically. For example, the instrument may detect electrochemicalspecies in the droplet and/or optically active molecules in the droplet(e.g., chromogenic molecules, fluorescent molecules and the like). Inaddition, the instrument may position a droplet at a nanopore layer andmeasure translocation of a tag or an analyte-specific binding member(e.g., an aptamer) though the nanopore layer.

The systems may further include memory with instructions that areexecuted on a processor included in the system (for example, included inthe instrument) for controlling the DMF electrodes and for controllingthe electrodes used for electrochemical detection or controlling the foroptical detection unit, and the like.

In certain embodiments, the analyte detection systems of the presentdisclosure may include an analyte detection device that includes aprocessor for executing a program with instructions for first activatingthe DMF electrodes for movement of sample droplets/bufferdroplets/reagent droplets and the like. The instructions may furtherinclude deactivating the DMF electrodes and measuring electrical signalsfrom a working electrode for detecting electrochemical species generatedin response to presence of an analyte in the sample. The system mayfurther include algorithms for normalizing the signal recorded from thechips, for example, to remove noise prior to determining concentrationof the analyte. The algorithms may include a calibration curve to assistin determining analyte concentration.

The systems disclosed herein may be used to process a sample droplet forgeneration of an electrical signal (e.g., from an electrochemicalspecies measured using working and reference electrodes or fromtranslocation of tags or analyte-specific binding members through ananopore) and/or an optical signal indicative of presence of the analytein the sample. The electrical and/or optical signal may generated byaction of an enzyme on a substrate in a clinical chemistry assay. Theelectrical signal may be generated by translocation of tags oranalyte-specific binding members through a nanopore. A sample may beprocessed utilizing one or more assay formats described in thisdisclosure.

The systems of the present disclosure may be used in a method forelectrochemical detection of an analyte in a sample. The method mayinclude (a) introducing the sample into a cartridge, the cartridgecomprising: a first substrate; a second substrate; a gap separating thefirst substrate from the second substrate; a plurality of electrodes togenerate electrical actuation forces on a liquid droplet; and anelectrochemical species sensing region comprising a working electrodeand a reference electrode; (b) actuating the plurality of electrodes toprovide a first liquid droplet comprising the analyte; (c) actuating theplurality of electrodes to provide a second liquid droplet comprising anenzyme specific for the analyte; (d) actuating the plurality ofelectrodes to merge the first and second droplets to create a mixture;(e) actuating the plurality of electrodes to move all or a portion ofthe mixture to the electrochemical sensing region; (f) detecting, viathe working and reference electrodes, an electrical signal of anelectrochemical species generated by action of the enzyme on theanalyte.

In some cases, the second liquid droplet may also include a redoxmediator. In some cases, the system may determine a concentration of theanalyte based on the electrical signal. In some cases, theelectrochemical sensing region is located in a capillary region in thecartridge.

In certain embodiments, the method may include (a) introducing thesample into a cartridge, the cartridge comprising: a first substrate; asecond substrate; a gap separating the second substrate from the firstsubstrate; a plurality of electrodes to generate electrical actuationforces on a liquid droplet; and an electrochemical species sensingregion comprising a working electrode and a reference electrode; (b)actuating the plurality of electrodes to provide a first liquid dropletcomprising the analyte; (c) actuating the plurality of electrodes toprovide a second liquid droplet comprising a solid substrate comprisinga first binding member that specifically binds to the analyte; (d)actuating the plurality of electrodes to merge the first and seconddroplets to create a mixture; (e) actuating the plurality of electrodesto merge all or a portion of the mixture with a third liquid dropletcomprising a second binding member that specifically binds to theanalyte; (f) holding the solid substrate in place while actuating theplurality of electrodes to remove any unbound analyte and/or secondbinding member; (g) actuating the plurality of electrodes to contact thesolid substrate with a substrate molecule for the enzyme conjugated tothe second binding member; and (h) detecting, via the working andreference electrodes, an electrical signal of an electrochemical speciesgenerated by action of the enzyme on the substrate molecule.

In some cases, the method may include moving a liquid droplet comprisingthe solid second substrate from step (f) to the electrochemical sensingregion prior to steps (g) and (h). In other cases, the method mayinclude moving a liquid droplet comprising the solid second substrateand enzyme substrate from step (g) to the electrochemical sensingregion.

In some cases, the second liquid droplet may also include a redoxmediator. In some cases, the system may determine a concentration of theanalyte based on the electrical signal. In some cases, theelectrochemical sensing region is located in a capillary region in thecartridge. As noted herein, the systems and instruments may perform twoor more separate assays using a multifunctional cartridge(s) or usingmultiple separate single assay cartridge.

In certain embodiments a method for performing analyte detection usingan instrument is disclosed. The method may include providing an analytedetection instrument comprising a cartridge interface for operableconnection to the one or more analyte detection cartridges; providing aplurality of cartridges having a plurality of electrodes to generateelectrical actuation forces on a liquid droplet: interfacing a firstcartridge with the instrument and detecting an analyte related signalfrom a droplet in a cartridge; and interfacing a second cartridge withthe instrument and detecting an analyte related signal from atag/analyte-specific binding member translocating through a pore of ananopore layer in the cartridge.

In certain cases, a system may include programming that allows it tointerface with a plurality of instruments, where each of the pluralitiesof instruments conducts a plurality of assays, where the plurality ofinstruments are different or same. For example, a system as depicted inFIG. 60A may include a control unit comprising a processor 413 operablyconnected to a plurality of devices 411, the plurality of devices mayeach include at least one slot (411 a-411 d) in which a cartridge may beinserted and operated upon by the processor. The devices 411 may beidentical and may provide means for increasing the number of samplesthat can be processed simultaneously. In certain embodiments, the systemmay include programming or may be upgraded to include programming thatallows it to interface with additional or alternate instruments as theybecome available. A system as depicted in FIG. 60B may include aprocessor 413 operably connected to a plurality of different devices 414a-414 d, the plurality of devices may each include at least one slot(412 a-411 d) in which a different type of cartridge may be inserted andoperated upon by the processor. For example, the first device may beconfigured for conducting an immunoassay, the second device may beconfigured for conducting an electrochemical assay, the third device maybe configured for conducting a hematology assay, and the like. In suchan embodiment, the device and the cartridge compatible with the devicemay include means for sample preparation and detection of an analyterelated signal (for sample analysis). The programming executed by theprocessor 413 may include instructions that are communicated to thedevices for preforming the steps required for conducting an assay, suchas, actuating DMF electrodes or generating SAW for sample preparationand controlling detection modules, such as, camera, microscope,electrochemical sensors, etc., for detecting a signal from the preparedsample. The system may additionally include algorithm for analyzingcollected data prior to providing assay results. The system may beequipped for wireless communication to provide assay results on a remotedevice connected wirelessly to the system. In certain cases, the remotedevice may receive the results in real time. In certain cases, a printermay also be connected to the system to provide a printout of the assayresults.

The modularity of the system depicted in FIGS. 60A and 60B allows foradding to or removing from the functionality of the system to provideflexibility to the consumer, e.g., at point-of-care facilities.

Analytes

A non-limiting list of analytes that may be analyzed by the methods,chips, instruments and methods presented herein include moleculespresent in a biological sample, such as, a blood sample (or a portionthereof, e.g., serum or plasma). Exemplary analytes of interest includenucleic acids, one or more of low density lipoprotein (LDL), highdensity lipoprotein (HDL), cholesterol, triglycerides, glucose,hemoglobin (Hb), HbA1c, albumin, microalbumin, total protein, sodium(Na⁺), potassium (K⁺), chloride (Cl⁻), carbon dioxide, oxygen,creatinine, calcium (Ca²⁺), blood urea nitrogen (BUN), pH, lactate,ketone bodies, alanine aminotransferase (ALT), aspartateaminotransferase (AST), alkaline phosphatase (ALP), bilirubin, ferritin,alcohol (blood alcohol), amphetamine, methamphetamine, cannabis,opiates, barbituarates, benzodiazapine, tricyclic acid, cocaine, andphencyclidine (PCP). Additional analytes that may be detected andoptionally measured using the DMF-electochemical/electrical/opticaldetection chips disclosed herein include one or more of the analytesdetected in the preceeding sections. Further examples of analytes thatmay be detected and measured using the methods, devices, and systems ofthe present disclosure include blood cells, flu virus, streptococcalbacteria, raus sarcoma virus, adenovirus, mononucleosis, tuberculosis,B-HCG, HIV, HCV, HBV, syphilis, herpes, troponin, BNP, CK-MB, myoglobin,D-dimer, PSA, TSH, T3, T4, FSH, LH, estradiol, testosterone, vitamin D,B12, and H. Pylori.

The sample in which the analyte is being detected may be any sampledisclosed herein, such as, a sample of blood, solid tissue, another bodyfluid, such as, urine, sputum, saliva, cerebrospinal fluid, as well as,environmental samples, such as, water, soil, food samples and the like.

10. EXAMPLES Example 1 Synthesis of Photocleavable 2-NitrobenzylSuccinimidyl/Maleimidyl Bifunctional Linker

Synthesis of Compound 2. Synthesis of the photocleavablesulfosuccinimidyl/maleimidyl linker is derived from Agasti, et al., J.Am. Chem. Soc., 134(45), 18499-18502, 2012. Briefly, starting material4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (0.334 mmol)is dissolved in dry dichloromethane (DCM) under argon atmosphere. Theflask is cooled to 0° C. by placing it in an ice bath. Compound2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (0.368 mmol) and trimethylamine (TEA) (0.835 mmol) are added tothe solution. The reaction mixture is stirred at 0° C. for 5 min andsubsequently N-(2-aminoethyl)maleimide trifluoroacetate salt (0.368mmol) is added. After stirring at 0° C. for 15 min, the reaction mixtureis allowed to rise to room temperature (RT) and further stirred for 18h. After dilution of the reaction mixture with DCM (45 ml), the organicphase is washed with water (2×), saturated NaCl solution (1×) and driedover sodium sulfate. The organic layer is concentrated under reducedpressure and purified by flash chromatography using a SiO₂ column(eluent: 100% DCM to 3% methanol in DCM, v/v). Compound 1 (0.024 mmol)is dissolved in anhydrous dimethylformamide (1 ml).N,N′-disulfosuccinimidyl carbonate (DSC) (0.071 mmol) and TEA (0.096mmol) are successively added to the solution. The reaction mixture isstirred at RT for 18 h. The reaction mixture is purified by directlyloading onto a C18 reverse phase column (eluent: 5% acetonitrile inwater to 95% acetonitrile in water, v/v). Starting material and otherchemicals used for the synthesis may be purchased from Sigma-Aldrich.

Example 2 Synthesis of Photocleavable Sulfosuccinimidyl/DBCO2-Nitrobenzyl Bifunctional Linker

Synthesis of Compound 4. Synthesis of the photocleavablesulfosuccinimidyl/dibenzocyclooctyl (DBCO) alkynyl linker is derivedfrom a similar procedure described in Agasti, et al., J. Am. Chem. Soc.,134(45), 18499-18502, 2012. Briefly, starting material4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (0.334 mmol)is dissolved in dry dichloromethane (DCM) under argon atmosphere. Theflask is cooled to 0° C. by placing it in an ice bath. Compound2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (0.368 mmol) and trimethylamine (TEA) (0.835 mmol) are added tothe solution. The reaction mixture is stirred at 0° C. for 5 min andsubsequently DBCO-amine (0.368 mmol) is added. After stirring at 0° C.for 15 min, the reaction mixture is allowed to rise to RT and furtherstirred for 18 h. After dilution of the reaction mixture with DCM (45ml), the organic phase is washed with water (2×), saturated NaClsolution (1×) and dried over sodium sulfate. The organic layer isconcentrated under reduced pressure and purified by flash chromatographyusing a SiO₂ column (eluent: 100% DCM to 3% methanol in DCM, v/v).Compound 3 (0.024 mmol) is dissolved in anhydrous dimethylformamide (1ml). N,N′-disulfosuccinimidyl carbonate (DSC) (0.071 mmol) and TEA(0.096 mmol) are successively added to the solution. The reactionmixture is stirred at RT for 18 h. The reaction mixture is purified bydirectly loading onto a C18 reverse phase column (eluent: 5%acetonitrile in water to 95% acetonitrile in water, v/v). Startingmaterial and other chemicals used for the synthesis may be purchasedfrom Sigma-Aldrich.

Example 3 Coupling and Photochemical Cleavage of Antibody-DNA ConjugateUsing Sulfosuccinimidyl/Maleimidyl 2-Nitrobenzyl Bifunctional Linker

Bioconjugation and Cleavage of Antibody and DNA. DNA molecules may beconjugated to antibodies using the following scheme. DNA may bethiolated at the 5′ terminus by replicating a DNA sequence in a PCRreaction using two PCR primers where one or both primers are labeledwith a 5′-thiol group. Labeled DNA (100 μM final concentration) isdissolved in 50 mM HEPES (pH=7.0) with stirring. Compound 2 (2 mM) isadded and the reaction is allowed to proceed at RT for 2 hours. Aftercoupling, excess unreacted maleimide groups are quenched with excessdithiothreitol (DTT). The conjugate is purified on a gel filtrationcolumn (Sephadex G-25) or by extensive dialysis at 4° C. in anappropriate conjugate storage buffer. Purified DNA-succinimidyl linker(50 μM final concentration) is dissolved in 100 mM PBS (pH=7.5) withstirring. Native antibody (50 μM final concentration) is added and thereaction is allowed to proceed at RT for 2 hours. The Ab-DNA conjugateis purified using a Sephadex column (Sephadex G25) operated with 100 mMPBS, pH 7.5, or BioGel P-30 gel filtration media.

The conjugate may be cleaved prior to nanopore detection by illuminatingwith a UV lamp at 365 nm. This example may also be used on DNAdendrimers using the same bioconjugation chemistry.

Example 4 Coupling and Photochemical Cleavage of Antibody-DNA ConjugateUsing Sulfosuccinimidyl/DBCO 2-Nitrobenzyl Bifunctional Linker

Bioconjugation and Cleavage of Antibody and DNA. DNA molecules may beconjugated to antibodies using the following scheme. DNA may be aminatedat the 5′ terminus by replicating a DNA sequence in a PCR reaction usingtwo PCR primers where one or both primers are labeled with a 5′-aminegroup. Labeled DNA (100 μM final concentration) is dissolved in 100 mMPBS (pH=7.5) with stirring. Compound 4 (2 mM final concentration) isadded and the reaction is allowed to proceed at RT for 2 hours. TheDNA-DBCO linker is purified on a gel filtration column (Sephadex G-25)or by extensive dialysis at 4° C. in an appropriate conjugate storagebuffer. Purified DNA-DBCO linker (50 μM final concentration) isdissolved in 50 mM Tris (pH=7.0) with stirring. Copper-free Clickchemistry is used to couple the DNA-DBCO linker to the antibody.Azido-labeled antibody (Kazane et al., Proc. Natl. Acad. Sci., 109(10),3731-3736, 2012) (25 μM final concentration) is added and the reactionis allowed to proceed at RT for 6-12 hours. The Ab-DNA conjugate ispurified using a Sephadex column (Sephadex G25) operated with 100 mMPBS, pH 7.5, or BioGel P-30 gel filtration media.

The conjugate may be cleaved prior to nanopore detection by illuminatingwith a UV lamp at 365 nm. This example may also be used on DNAdendrimers using the same bioconjugation chemistry.

Example 5 Nanoparticle-Antibody Conjugates for Digital Immunoassays(Nanopore Counting)

This example describes covalent conjugation of an antibody to 26 nmcarboxylated polystyrene nanoparticles (NP, PC02N), such as those whichcan be obtained from Bangs Labs (Fishers, Ind., USA). The 26 nm NPs havea surface charge of 528.7 μeq/g and a parking area of 68.4 sq·Å/group(per manufacturer information).

Activation of carboxyl-polystyrene nanoparticles: 1.0 mL (100 mg/mL) of26 nm carboxylated-NP is washed with 10 mL of 0.1M MES(2-[N-morpholino]ethane sulfonic acid, pH 4.5-5.0. After the wash, thepellets are resuspended in 100 mL of 0.1M MES pH 4.5-5.0 for a 1.0 mg/mLNP concentration (0.1% solids). 10.0 mL nanoparticle suspension (10 mgNP, 5.28 μeq carboxyl) is transferred to a vial and reacted with 10 μL(5.28 moles, 1.0 equiv/CO₂H eq) of a freshly prepared 10 mg/mL EDCsolution in water (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride) and 17 μL (7.93 μmoles, 1.5 equiv/1 equiv EDC) of a 10mg/mL solution of sulfo-NHS solution in water(N-hydroxysulfosuccinimide, Sigma, Cat #56485) at room temperature for15 min with continuous mixing. The reacted suspension is centrifuged at6,500 g and the solution is discarded. The pellet is washed with 20 mLof 20 mM PBS/5 mM EDTA pH 7.5 and spun down by centrifugation at 6,500g. The supernatant is removed. The succinimide-activated carboxyl-NPpellet is resuspended in 50 mM PBS pH 7.5 and 9.8 μL (52.8 nmoles, 0.01equiv/1 CO₂H eq) of a 1.0 mg/mL pyridine dithioethylamine solution inwater is immediately added and allowed to react with continuous stirringfor 2-4h at room temperature. The pyridyl-derivatized carboxyl-NP iswashed with 10 mL of 20 mM PBS/5 mM EDTA pH 7.5 and resuspended in 10.0mL of the same buffer. The nanoparticle concentration is determinedusing UV-Vis spectroscopy (600 nm, scatter) using a carboxyl-NPcalibration curve. Pyridyl-ligand loading on the NP is determined byreducing a defined amount of NP with 10 mM TCEP or DTT, removing thereducing agent by centrifugation, resuspending the pyridyl-activated NPpellet in PBS/EDTA pH T2 and reacting with the Ellman reagent (measureA412 of the supernatant), The activated NP is stored at 0.1° C. if notused on the same day for antibody conjugation.

A range of EDC/NHS and pyridine dithioethyl amine molar inputs areevaluated to determine the desired stoichiometry for preparing distinctantibody-nanoparticle conjugates. Reaction parameters (pH, temperature,time) are assessed to achieve the desired NP activation outcome.

Antibody reduction: 1.0 mL of a 10 mg/mL antibody solution (10 mg) ismixed well with 38 μL of a freshly prepared 30 mg/mL 2-MEA solution (10mM reaction concentration) (2-mercaptoethylamine hydrochloride), thencapped and placed at 37° C. for 90 min. The solution is brought to roomtemperature and the excess 2-MEA is removed with a desalting column,pre-equilibrated in 20 mM PBS/5 mM EDTA pH 7.5. The concentration of thereduced antibody is determine using UV-Vis absorbance at A280 (proteinabsorbance) and A320 (scatter correction). The number of free thiols isdetermined using the Ellman test. The conditions are optimized as neededto generate 2 or 4 free thiols (Cys in the antibody hinge region). Thereduced antibody is used immediately for coupling to pyridyl-derivatizedcarboxyl-NP.

Coupling of reduced antibody to activated-nanoparticle: Assumptionsmade: (1) antibody parking area is 45 nm²; (2) 2.6 nm nanoparticlesurface area is 2,120 nm²; (3) 47 antibody molecules theoretically fiton the surface of a 26 nm NP.

Procedure: To 10 mL 1t) mg) of a 0.1% solution of pyridyl-activatedcarboxy-nanoparticles in 20 mM PBS/5 mM EDTA (pH 7.5), 0.10 mg (0.66nmoles, 0.10 mL) of the reduced antibody is added at 1.0 mg/mL in thesame EDTA containing buffer. The mixture is allowed to react at roomtemperature with mixing for 2h, centrifuged to remove unbound molecules,and aspirated. The pellet is washed with 10 mL of PBS pH 7.2,centrifuged, and aspirated. The antibody-NP conjugate is suspended in10.0 mL of PBS pH 7.2. The conjugate NP concentration (% solids) isdetermined using UV-Vis spectroscopy (600 nm). The particle conjugate isexamined by SEM and the size/charge distribution is determined using theZetaSizer. Size exclusion chromatography can be used to isolate distinctconjugates from a potential distribution of conjugate population.Antibody-to-NP incorporation ratio can be determined by flow cytometryusing fluorescently labelled antigen conjugate or using a Micro BCA(uBCA) assay. A range of antibody-to-NP molar inputs can be evaluated,along with conjugation temperature and pH to generate a homogenouspopulation of distinct conjugates (i.e., NP incorporation ratio of 2 or4).

Nanopore Counting Immunoassay

The scheme above illustrates the nanopore counting assay utilizing thereduced antibody-activated nanoparticle conjugate whose preparation isdescribed above. The immune complex formed in the course of theimmunoassay can be cleaved by reduction of the disulfide bond linker toform the free antibody-analyte-antibody complex and free nanoparticletag, which permits the nanoparticle tag to be counted upon passagethrough the nanopore.

Example 6 Synthesis of CPSP Conjugates

A. CPSP Antibody Conjugate.

3-(9-((4-Oxo-4-(perfluorophenoxy)butyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonate(2): A 25 mL round bottom flask equipped with a magnetic stirrer and anitrogen inlet was charged with3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonate(CPSP) (1) (1 mmol), pyridine (5 mmol) and dimethylformamide (10 mL).The solution was cooled in an ice bath and pentafluorophenyltrifluoroacetate (1.3 mmol) was added dropwise. The ice bath was removedand the reaction was stirred at room temperature for 3 hours. Thevolatile components were removed from the reaction in vacuo and theresidue was taken up in methanol and purified by reverse phase HPLC togive the title compound.

CPSP antibody conjugate (3): A solution of 2 (1 μL of a 10 mM solutionin DMSO) was added to an antibody solution (100 μL of a 10 μM solutionin water) and aqueous sodium bicarbonate (10 μL of a 1M solution). Theresulting mixture was stirred at room temperature for 4 hours.Purification of the product was achieved on a spin column to give theCPSP antibody conjugate 3. The value of “n” varies in anantibody-dependent fashion. The incorporation can be controlled to someextent by raising or lowering the active ester concentration (i.e.,compounds 2, 5, 9 and 13) and/or by raising or lowering the pH duringthe reaction, but always results in a distribution of incorporationvalues. The average incorporation ration (“I.R.”) is determinedexperimentally after the reaction. Typically, “n” is any value between 1and 10.

B. CPSP Antibody Conjugate with Spacer.

3-(9-((4-((5-Carboxypentyl)amino)-4-oxobutyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonate(4): A 25 mL round bottom flask equipped with a magnetic stirrer and anitrogen inlet was charged with3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonateCPSP (1) (1 mmol), pyridine (5 mmol) and dimethylformamide (10 mL). Thesolution was cooled in an ice bath and pentafluorophenyltrifluoroacetate (1.3 mmol) was added dropwise. The ice bath was removedand the reaction was stirred at room temperature for 3 hours.6-Aminocaproic acid (1.3 mmol) was then added to the reaction in smallportions followed by N,N-diisopropylethylamine (5 mmol), and thereaction was stirred for 1 hour at room temperature. After this time,the volatile components were removed from the reaction in vacuo and theresidue was purified by reverse phase HPLC to give the title compound.

3-(9-((4-Oxo-4-((6-oxo-6-(perfluorophenoxy)hexyl)amino)butyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonate (5): A 25 mL round bottom flaskequipped with a magnetic stirrer and a nitrogen inlet was charged with 4(1 mmol), pyridine (5 mmol) and dimethylformamide (10 mL). The solutionwas cooled in an ice bath and pentafluorophenyl trifluoroacetate (1.3mmol) was added dropwise. The ice bath was removed and the reaction wasstirred at room temperature for 3 hours. After this time, the volatilecomponents were removed from the reaction under a stream of nitrogen andthe residue was purified by reverse phase HPLC to give the titlecompound.

CPSP antibody conjugate with spacer (6): A solution of 5 (1 μL of a 10mM solution in DMSO) was added to an antibody solution (100 μL of a 10μM solution in water) and aqueous sodium bicarbonate (10 μL of a 1Msolution). The resulting mixture was stirred at room temperature for 4hours. Purification of the product was achieved on a spin column to givethe CPSP antibody conjugate with spacer 6. Typically, “n” is any valuebetween 1 and 10.

C. CPSP Oligonucleotide-Antibody Conjugate.

9-((3-Carboxypropyl)(tosyl)carbamoyl)-10-(prop-2-yn-1-yl)acridin-10-ium(8): A 100 mL round bottom flask equipped with a magnetic stirrer and anitrogen inlet was charged with propargyl alcohol (10 mmol),2,6-di-tert-butylpyridine (10 mmol) and methylene chloride (50 mL) andcooled to −20° C. Triflic anhydride was then added dropwise to thesolution and the reaction was stirred for 2 hours at −20° C. Pentane (25mL) was added to the reaction and the resulting precipitated salts wereseparated by filtration. The volatile components were evaporated invacuo and the residue was redissolved in methylene chloride (25 mL) in a100 mL round bottom flask. 4-(N-Tosylacridine-9-carboxamido)butanoicacid (CP-acridine) (7) (1 mmol) was added in small portions and thereaction was stirred at room temperature for 18 hours. The volatilecomponents were evaporated in vacuo and the residue was taken up inmethanol (5 mL) and purified by reverse phase HPLC to give the titlecompound.

9-((4-oxo-4-(perfluorophenoxy)butyl)(tosyl)carbamoyl)-10-(prop-2-yn-1-yl)acridin-10-ium(9): A 25 mL round bottom flask equipped with a magnetic stirrer and anitrogen inlet was charged with 8 (1 mmol), pyridine (5 mmol) anddimethylformamide (10 mL). The solution was cooled in an ice bath andpentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. Theice bath was removed and the reaction was stirred at room temperaturefor 3 hours. The volatile components were removed from the reaction invacuo and the residue was taken up in methanol and purified by reversephase HPLC to give the title compound.

CPSP antibody conjugate (10): A solution of 9 (1 μL of a 10 mM solutionin DMSO) was added to an antibody solution (100 μL of a 10 μM solutionin water) and aqueous sodium bicarbonate (10 μL of a 1M solution). Theresulting mixture was stirred at room temperature for 4 hours.Purification of the product was achieved on a spin column to give theCPSP antibody conjugate 10. Typically, “n” is any value between 1 and10.

CPSP oligonucleotide-antibody conjugate (11): A mixture of an oligoazide(10 nmol in 5 μL water), CPSP antibody conjugate 10 (10 nmol in 10 μLwater) and a freshly prepared 0.1 M “click solution” (3 μL—see below)was shaken at room temperature for 4 hours. The reaction was dilutedwith 0.3M sodium acetate (100 μL) and the DNA conjugate was precipitatedby adding EtOH (1 mL). The supernatant was removed and the residue waswashed 2× with cold EtOH (2×1 mL). The residue was taken up in water (20μL) and the solution of the CPSP oligonucleotide-antibody conjugate 11was used without further purification. Typically, “n” is any valuebetween 1 and 10.

“click solution”: CuBr (1 mg) was dissolved in 70 μL DMSO/t-BuOH 3:1 toform a 0.1 M solution. (This solution must be freshly prepared andcannot be stored.) Tris(benzyltriazolylmethyl)amine (TBTA) (54 mg) wasdissolved in 1 mL DMSO/t-BuOH 3:1 to form a 0.1 M solution. (Thissolution can be stored at −20° C.) 1 volume of the 0.1 M CuBr solutionwas added to 2 volumes of the 0.1 M TBTA solution to provide a “clicksolution.”

D. CPSP Oligonucleotide-Antibody Conjugate with Spacer.

9-((4-((5-carboxypentyl)amino)-4-oxobutyl)(tosyl)carbamoyl)-10-(prop-2-yn-1-yl)acridin-10-ium(12): A 25 mL round bottom flask equipped with a magnetic stirrer and anitrogen inlet was charged with 8 (1 mmol), pyridine (5 mmol) anddimethylformamide (10 mL). The solution was cooled in an ice bath andpentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. Theice bath was removed and the reaction was stirred at room temperaturefor 3 hours. 6-Aminocaproic acid (1.3 mmol) was then added to thereaction in small portions followed by N,N-diisopropylethylamine (5mmol), and the reaction was stirred for 1 hour at room temperature.After this time, the volatile components were removed from the reactionin vacuo and the residue was purified by reverse phase HPLC to give thetitle compound.

9-((4-oxo-4-((6-oxo-6-(perfluorophenoxy)hexyl)amino)butyl)(tosyl)carbamoyl)-10-(prop-2-yn-1-yl)acridin-10-ium(13): A 25 mL round bottom flask equipped with a magnetic stirrer and anitrogen inlet was charged with 12 (1 mmol), pyridine (5 mmol) anddimethylformamide (10 mL). The solution was cooled in an ice bath andpentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. Theice bath was removed and the reaction was stirred at room temperaturefor 3 hours. After this time, the volatile components were removed fromthe reaction under a stream of nitrogen and the residue was purified byreverse phase HPLC to give the title compound.

CPSP antibody conjugate with spacer (14): A solution of 13 (1 μL of a 10mM solution in DMSO) was added to an antibody solution (100 μL of a 10μM solution in water) and aqueous sodium bicarbonate (10 μL of a 1Msolution). This was stirred at room temperature for 4 hours.Purification of the product was achieved on a spin column to give theCPSP antibody conjugate with spacer 14. Typically, “n” is any valuebetween 1 and 10.

CPSP oligonucleotide-antibody conjugate (15): A mixture of an oligoazide(e.g., such as is commercially available) (10 nmol in 5 μL water), CPSPantibody conjugate with spacer 14 (10 nmol in 10 μL water) and a freshlyprepared 0.1 M “click solution” (3 μL—see Example 6.C) was shaken atroom temperature for 4 hours. Typically, “n” is any value between 1 and10. The reaction was diluted with 0.3M sodium acetate (100 μL) and theDNA conjugate was precipitated by adding EtOH (1 mL). The supernatantwas removed and the residue was washed 2× with cold EtOH (2×1 mL). Theresidue was taken up in water (20 μL) and the solution of the CPSPoligonucleotide-antibody conjugate with spacer 15 was used withoutfurther purification.

Cleavage of CPSP antibody conjugates with or without spacer and CPSPoligonucleotide-antibody conjugate with or without spacer. The CPSPantibody conjugates with or without spacer and CPSPoligonucleotide-antibody conjugate with or without spacer, as described,are cleaved or “triggered” using a basic hydrogen peroxide solution. Inthe ARCHITECT® system, the excited state acridone intermediate producesa photon, which is measured. In addition, the cleavage products are anacridone and a sulfonamide. The conjugates of Examples 6.A-D are usedwith the disclosed device by counting the acridone and/or sulfonamidemolecules.

E. CPSP Oligonucleotide Conjugate with No Antibody.

CPSP oligonucleotide conjugate with no antibody (16): A mixture of anoligoazide (e.g., such as is commercially available) (10 nmol in 5 μLwater), propargyl-CPSP 8 (10 nmol in 10 μL water) and a freshly prepared0.1 M “click solution” (3 μL—see Example 6.C) can be shaken at roomtemperature for 4 hours. The reaction can be diluted with 0.3M sodiumacetate (100 μL) and the DNA conjugate precipitated by adding EtOH (1mL). The supernatant can be removed and the residue washed 2× with coldEtOH (2×1 mL). The residue can be taken up in water (20 μL) and thesolution of the CPSP oligonucleotide-antibody conjugate with spacer 16can be used without further purification.

Example 7 Fabrication of Low-Cost DMF Bottom Chip

Low-cost flexible DMF chips were fabricated using roll-to-roll (R2R)flexographic printing combined with a wet lift-off process for electrodepatterning by using the process described in Lo C-Y et al.,Microelectronic Engineering 86 (2009) 979-983 with some modifications. Aschematic of the fabrication process is depicted in FIG. 10. A roll ofMELINEX® ST506 polyethylene terephthalate (PET) 5.0 mil substrate (1)was used as the starting material for DMF electrode printing. A layer ofyellow ink (Sun Chemical) was flexo-printed (2) on the PET substrateusing a 1.14 mm thick printing plate (Flint MCO3) at a rate of 10m/minute using an ink transfer volume of 3.8 ml/m² on an Anilox rollerassembly. A negative image of the DMF electrode pattern results from theflexo printing step (3). Prior to metal deposition, the ink was driedtwo times in a hot air oven (2×100° C.). An EVA R2R Metal Evaporator wasused to deposit a layer of silver metal onto the printed PET substrateto form a uniform coating of silver at a thickness of 80 nm (4). Themetalized ink-film substrate (5) was subjected to a wet lift-off processusing a combination of acetone plus ultrasound in a sonication bath at aspeed of 1 m/minute (6). This chemical/physical treatment allows thesilver-ink layer to dissolve, while keeping the silver-only layerintact. Removal of the ink-silver layer resulted in a DMF printedelectrode pattern consisting of 80 actuation electrodes (2.25×2.25 mm)with either 50 or 140 μm electrode gap spacing (7). As a QC check, atotal of 80-90 random chips from a single roll were visually inspectedfor electrode gap spacing and connector lead width variation. Typicalyields of chips, determined to have acceptable gap specifications, wereclose to 100%. A single fabricated flexible chip is depicted in FIG. 11.The fabricated flexible chip measures 3″×2″ and includes electrodes,reservoirs, contact pads and leads.

A dielectric coating was applied to the electrodes and reservoirs byusing either rotary screen printing or Gravure printing. For rotaryscreen printing, Henkel EDAC PF-455B was used as a dielectric coating byprinting with a Gallus NF (400 L) screen at a printing speed of 2m/minute and a UV curing rate of 50%. Typical dielectric thickness was10-15 μm. For Gravure printing, cylinders were designed to print ahigh-viscosity dielectric ink, such as IPD-350 (Inkron), at a speed of 2m/minute using an ink volume of 50 ml/m². Typical dielectric thicknessfor Gravure printing was 7-8 μm. A final hydrophobic layer was printedusing either Millidyne Avalon 87 or Cytonix Fluoropel PFC 804 UC coatingwith Gravure cylinders (140-180 L) and a printing speed of 8 m/minute,followed by four successive oven drying steps (4×140° C.). Typicalhydrophobic thickness was 40-100 nm.

Alternatively, for small batches of individual chips, the dielectric andhydrophobic coatings may be applied using chemical vapor deposition(CVD) and spin coating, respectively.

Example 8 Functional Testing of Low-Cost DMF Chip

A 3″×2″ PET-based DMF bottom chip manufactured as outlined in Example 7above was tested for actuation capability. FIG. 12 depicts a 3″×2″PET-based DMF chip (1) over which a 0.7 mm thick glass substrate (3) ispositioned. The glass substrate (3) includes a transparent indium tinoxide (ITO) electrode on a lower surface of the glass substrate and aTeflon coating over the ITO electrode. The DMF chip includes 80 silveractuation electrodes with a straight edge electrode design and a 50 μmgap between electrodes, along with 8 buffer reservoirs (see Example 7above).

The bottom electrodes were coated with a layer of dielectric Parylene-C(6-7 μm thick) and a final coating of Teflon (50 nm thick) by CVD andspin-coating, respectively. Approximately 50 μL of PBS buffer with 0.1%surfactant (2) was pipetted into four adjacent reservoirs on the bottomDMF chip. Droplet sizes ranged from 700-1,500 nL (one or two droplets)and were checked for both vertical and horizontal lateral movement (4),in addition to circular sweep patterns necessary for mixing. Dropletactuation was achieved using a voltage of 90 V_(rms). Approximately 90%of the actuation electrodes on the chip were tested and found to befully functional.

Example 9 TSH Immunoassay on Low-Cost DMF Chip

The 3′×2″ PET-based DMF chip overlayed with the glass substrate asdescribed in Example 8 above, was tested for its ability to carry out athyroid stimulating hormone (TSH) immunoassay, using chemiluminescencedetection. Mock samples included TSH calibrator material spiked intotris buffered saline (TBS) buffer containing a blocking agent and asurfactant. Three samples were tested—0, 4, 40 μIU/ml. 2 μL of anti-betaTSH capture antibody, coated on 5 μm magnetic microparticles (3×10⁸particles/ml), was dispensed from the microparticle reservoir into themiddle of the DMF electrode array. The magnetic microparticles wereseparated from the buffer by engaging a neodymium magnet bar (3 in.×½in.×¼ in. thick, relative permeability μ_(r)=1.05, remnant fieldstrength B_(r)=1.32 T) under the DMF chip (FIG. 13A). 5 μL of sample wasmoved to the microparticle slug, followed by mixing the microparticlesuspension (FIG. 13B) over a four-electrode square configuration for 5minutes. The microparticles were separated from the sample by themagnet, and the supernatant was moved to a waste reservoir (FIGS. 13Cand 13D). 2 μL of 1 μg/mL anti-TSH detection antibody conjugated tohorseradish peroxidase (HRP) was moved to the microparticle slug andmixed for 2 minutes. The microparticles were separated by the magnet,and the supernatant was moved to the waste reservoir. The microparticlescontaining the immunoassay sandwich complex were washed a total of fourtimes with 4×2 μL of PBS wash buffer containing 0.1% surfactant. Washbuffer from each wash step was moved to waste after the step wascompleted. Chemiluminescent substrate consisted of 1 μL of SuperSignalH₂O₂ and 1 μL luminol (ThermoFisher Scientific), which was moved to themicroparticle slug, followed by mixing for 6 minutes. Chemiluminescentsignal was measured at 427 nm emission (347 nm excitation) using anintegrated Hamamatsu H10682-110 PMT with a 5 V DC source. Adose-response curve was plotted against relative luminescence (see FIG.13E).

Example 10 Nanopore Module Fabrication

A nanopore module was fabricated using standard soft lithographyfabrication methods coupled with integration of a commercially availablesilicon nitride (SiN_(x)) membrane embedded in a TEM window (Norcada).The module consisted of four separate layers of PDMS—a top and bottomPDMS substrate containing the transfer microchannels, and two optionalintermediate PDMS layers to seal the TEM window.

SU8 Master Mold Fabrication: A clean, dry glass substrate was spincoatedwith photoresist (SU8-50) to a desired thickness. Areas of the coatedsubstrate were then selectively exposed to near-UV light using aphotomask. The mask exposes photoresist to UV light only in regionswhere the transfer microchannel and reservoir shapes are to remain.Exposure was followed by a bake to cross-link regions of photoresistthat were exposed. An SU8 developer was then used to remove remaining,unexposed photoresist from the substrate. The final product is a mastermold—a glass substrate with patterned transfer microchannels andreservoirs of hard photoresist.

Intermediate PDMS Layer Fabrication: For fabricating the intermediatePDMS layers, a solution containing PDMS monomer and its curing agent(Sylgard 184 silicone elastomer) in the ratio of 7:1 PDMS monomer:curingagent was spincoated on a glass slide, followed by heating on a hotplate for 30 minutes at 70° C. The PDMS layers were peeled off the glasssubstrate and 1.25 mm cut-out was punched through the PDMS layers toprovide an opening allowing access to the TEM window. Surface of thePDMS layers was made hydrophilic by plasma treating for 30 seconds usinga corona treater at a distance of 8 mm. A second plasma treatment (5seconds) was used to treat the surface of the PDMS layers and TEM windowbefore bonding the SiN_(x) TEM window between the two intermediate PDMSlayers.

Top and Bottom PDMS Fabrication: The top and bottom PDMS substratescontaining microchannels were fabricated, as shown in FIG. 14B, bymixing PDMS monomer and curing agent in a ratio of 7:1 PDMSmonomer:curing agent and pouring over glass containing the SU8 patternedmold (6) patterned with the transfer microchannels and reservoirs (seeSU8 Master Mold Fabrication described above). The microchannels measuredapproximately 110 to 135 μm in width and 50 μm in depth. After degassingfor 15 minutes, the SU8 mold was heated on a hot plate for 60 minutes at70° C. (7). After curing, the PDMS substrates were peeled off the SU8mold (8) and cut to yield rectangular PDMS substrates having anapproximate dimension of 30 mm length×20 mm width×3 mm depth. Accessholes (1.25 mm in diameter) were punched through the PDMS substrates toallow subsequent insertion of electrodes into the microchannels. Thefinal assembly is shown in FIG. 14A and includes from bottom to top,bottom PDMS substrate containing one microchannel (1), a firstintermediate PDMS layer (2) containing a cut-out positioned over themicrochannel, the SiN_(x) membrane in TEM window (3), a secondintermediate PDMS layer (4) also containing a cut-out, and a top PDMSsubstrate (5) containing a second microchannel.

Alignment of Top and Bottom PDMS Substrates: A PDMS bottom substrate(prepared as outlined in “Top and Bottom PDMS Fabrication,” above) wasplasma treated for 30 seconds, followed by bonding of a firstintermediate PDMS layer (prepared as outlined in “Intermediate PDMSLayer Fabrication,” above) onto the PDMS bottom substrate. Similarly, aPDMS top substrate was plasma treated for 30 seconds, followed bybonding of a second intermediate PDMS layer onto the PDMS top substrate.The cut-outs in the intermediate layers were aligned with themicrochannels. Both top and bottom PDMS pieces were oxygen plasmatreated for 30 seconds, followed by placement of the SiN_(x) membranewindow in between the top and bottom pieces and aligned with thecut-outs in the intermediate PDMS layers. The top piece aligned with theSiN_(x) membrane aligned with the bottom piece were pressed togetheruntil all air bubbles were released. The final nanopore PDMS assemblywas heated on a hot plate for at 100° C. for 30 minutes and plasmatreated for 5 minutes. The final module assembly, shown in FIG. 14C, (9a) contained two channels (one straight and one “L-shaped” channel),each ending in a reservoir for a solution (e.g., a buffer). The TEMwindow containing the SiN_(x) membrane is positioned at the intersectionof the two perpendicular microchannels (FIG. 14C, 9 b).

Example 11 Nanopore Fabrication

Nanopore fabrication was accomplished by subjecting a SiN_(x) TEMwindow, housed between two PDMS layers, to a potential bias untildielectric breakdown occurred, thereby opening up a small-diameter holein the membrane. This allows for in situ formation of a pore within themicrofluidic device, prior to detection of analytes. Nanopore formationby dielectric breakdown has been previously shown to be useful for rapidfabrication of small diameter pores in solid-state dielectric membranes(H. Kwok, K. Briggs, V. Tabard-Cossa, PLoS-One, 9(3), 2014).

SiN_(x) membrane commercially available as transmission electronmicroscope (TEM) windows (Norcada) were embedded in the assembled PDMSmodule as outlined in Example 10 above) and were used to generate thenanopore. The perpendicular microchannel junction exposed a crosssectional area (50 μm×50 μm) of the SiN_(x) TEM window to a saltsolution (1 M KCl) disposed on opposite sides of the membrane (cis andtrans). Ag/AgCl electrodes were placed into each microchannelapproximately 3 mm from the center of the SiN_(x) TEM window into holespunched through the PDMS substrate. A syringe containing a blunt needlewas used to fill both cis and trans microchannels by adding ethanol tothe two reservoirs until liquid was observed emerging from the channelopenings on the module edge. The resistance was measured to check forproper sealing and to ensure the TEM-SiN_(x) membrane was intact. Aresistance on the order of MΩ indicated good sealing and a membrane thatwas intact and undamaged. The ethanol was flushed out of themicrochannel with deionized water, and replaced with a 1 M KCl solutionby injecting into the two reservoirs. The resistance was measured againto check for proper sealing.

A constant voltage of 4.4 V was applied to the membrane assembly and theleakage current was monitored in real-time. The leakage current measuredin real-time is plotted in FIG. 15A. FIG. 15A shows the leakage current(1) prior to nanopore creation. A threshold value of >5 nA was used asthe cut-off value, i.e.—to signify pore creation. After approximately 10minutes, an increase in leakage current was observed (2). The voltagewas turned off immediately following the detection of increase inleakage current. The diameter of the created pore was 6.9 nm, asdetermined by the following relationship:

$G = {\sigma\left( {\frac{4L}{\pi\; d^{2}} + \frac{1}{d}} \right)}^{- 1}$

where G=conductance, σ=bulk conductivity (12.35 S/m measured for KCl),L=thickness of the membrane (10 nm), d=pore diameter (S. Kowalczyk, A.Grosberg, Y. Rabin, C. Dekker, Nanotech., 22, 2011).

After pore creation, a current-voltage (I-V) curve (see FIG. 15B) wasused to verify that the nanopore displayed ohmic behavior, indicatingthe nanopore was symmetrical in shape and the resistance was independentof the applied voltage or current. The same 1 M KCl solution was usedfor both pore fabrication and I-V curves.

Example 12 Dry Microchannel Filling

The capillary conduit contained in the assembled PDMS module (i.e., theintegrated device including a DMF module and a nanopore module) wastested for its ability to spontaneously fill high-salt solutions fromthe DMF electrode assembly (FIGS. 16A-16C). Filling was achieved viaspontaneous capillary flow (SCF). The nanopore membrane was not includedin order to allow for better visualization of the microchannels. Withreference to FIG. 16A, a glass DMF chip (3″×2″×0.0276″) containing 80actuation electrodes (1) (2.25 mm×2.25 mm, Cr-200 nm thickness) was usedto move a droplet (2) of 3.6 M LiCl, 0.05% Brij 35 and blue dye (to aidwith visualization). The PDMS module (3) contained two openings facingthe DMF electrode array (4), two reservoirs (5) and twomicrochannels—one straight channel (6) and one L-shaped channel (7). Themodule assembly was placed on the DMF glass surface so that the twochannel openings faced the interior of the DMF electrode array. Since atop grounding electrode chip was not used, droplet movement was achievedby using co-planar bottom electrodes to generate the driving potential.

A 10 μL droplet of blue-colored LiCl salt solution was placed on anelectrode in the middle of the DMF electrode array. A voltage of 100V_(rms) (10 kHz) was used to move the droplet to the transfer electrodeadjacent to the straight microchannel opening. As shown in FIG. 16B,after the droplet contacted the PDMS surface (8), the time required tofill the 130 μm diameter straight channel (9) and reach the reservoirwas measured. As shown in FIG. 16C, after approximately 30 seconds, thevolume of the droplet was visibly smaller (10) and the channel was halffilled (11). A total time of 53 seconds was required to fill the entiredry microchannel (130 μm diameter).

Wet Microchannel Filling: A 10 μL droplet of blue-colored LiCl saltsolution was placed on an electrode in the middle of the DMF electrodearray. A voltage of 100 V_(rms) (10 kHz) was used to move the droplet tothe transfer electrode adjacent to the straight microchannel opening.The channel was pre-filled with ethanol to mimic a pre-wetted channel.After the droplet contacted the PDMS surface, a time of <1 second wasrequired to fill the channel up to the reservoir. This was significantlyfaster than the dry channel, suggesting pre-wetting with a hydrophilicsolution enhances microchannel fill rates.

Example 13 DMF Droplet Transfer in Integrated Silicon NP Device

In addition to flexible substrates, such as PDMS, rigid substrates (e.g.silicon) may be used to fabricate the nanopore module. FIG. 17 shows adigital microfluidics (DMF) chip (1), containing actuation electrodes(4), from which droplets are transferred to a silicon microfluidic chipcontaining a nanopore sensor (2). Droplets are transferred between thetwo component chips by access ports (3) in the top surface of themicrofluidic chips containing the nanopore sensor. Access ports areconnected to the nanopore sensor (5) by microfluidic channels (6).Droplets are moved from the access ports, through the microfluidicchannels by capillary forces, and movement may be aided by a passivepaper pump fabricated from an array of micropillars (7) (FIG. 18). Thepassive pumps may also remove fluid from the microchannels, enablingdifferent fluidic solutions to be used sequentially withoutcontamination (for example, between solutions for nanopore formation andnanopore sensing).

Fabrication of the silicon nanopore module may include using standardCMOS photolithography and etching processes. FIG. 18 shows an example ofa silicon nanopore module design, where the approximate die size is 10mm×10 mm, with a frontside channel (cis) and a backside channel (trans)for filling the nanopore buffer(s). The frontside channel has a widthand depth of 30 μm, and is 11 mm long. The backside channel has a widthof 50 μm, a depth of 200 μm, and is 11 mm long. The micropillardimensions are 30 μm pillar diameter, spacing of 30 μm and depth of 200μm.

The DMF and nanopore module may be joined using an interface fabricatedfrom molded plastic or by direct bonding (FIGS. 19 and 20). A dropletpositioned on an electrode (4) within the DMF chip aligned with anaccess port is transferred by capillary forces, facilitated by theinterposer (7). Alternatively, the top electrode (8) the DMF chip may bemodified to further facilitate this process by introducing holesconnecting the actuation electrodes (4) with the interposer (8) (FIG.20).

Example 14 Droplet Transfer Between DMF and Nanopore Modules byCapillary Forces

The ability to move high-salt translocation buffer from a DMF chip to amodule containing a suitable nanopore membrane was tested in a siliconmicrofluidic chip. A serpentine microchannel was tested for its abilityto passively move a droplet of 1 M KCl (pH=8) using spontaneouscapillary flow (SCF) as the sole driving force. The entire microchannelwas fabricated in silicon and served as a model for fluidic transfer ina CMOS-based silicon environment. The serpentine microchannel wasdesigned to have two access ports (for fluidic loading). The channeldimensions measured 160 μm in diameter, with an approximate length of2.5 cm. Droplets of a solution suitable for formation of nanopores bydielectric breakdown were demonstrated to fill the silicon microfluidicstructure using passive capillary forces.

With reference to FIG. 21, individual droplets of 1M KCl solution(pH=8.0) were placed in one of the inlet ports (1) connecting to atransport microfluidic channel (2), leading to a serpentine channel 2.5cm in length (3). The channel terminated (4) at a port (not shown)exposed to atmospheric pressure. A magnified image of the serpentinechannel is shown in FIG. 22. Capillary filling was monitored using asCMOS camera fitted to an optical microscope. Deposition of the saltsolution into the inlet port resulted in spontaneous filling of themicrochannel by passive capillary forces at a rate of several mm/second,thereby demonstrating the capability to transfer fluid in themicrochannel to a nanopore membrane.

As a further test of transfer rate, the channel was emptied of the KClsolution and dried under a stream of nitrogen. Further droplets of 1MKCl solution (pH=8.0) were placed in the inlet port of the driedmicrochannel and capillary filling was monitored using and opticalmicroscope. Faster fill rates were observed, compared to the “dry”channel (i.e., compared to the first time the KCl solution wasintroduced into the channel), thereby showing that pre-filling of thesilicon microchannel with a hydrophilic solution enhanced subsequentfluidic filling.

Example 15 Fabrication of Integrated Nanopore Sensor with FluidicMicrochannels

An integrated nanopore sensor within fluidic microchannels is fabricatedusing photolithography and etching processes to modify asilicon-on-oxide (SOI) wafer (FIGS. 23A-23B).

The SOI wafer (1) is subjected to photolithography and etching (2) toproduce a structure suitable for the movement of small fluidic volumes(3) with dimensions of 30 μm width and 10-30 μm channel depth.

A silicon nitride (SiN) material (5) is deposited onto the patterned SOIwafer by evaporation (4).

A layer of oxide material (7) is deposited over the silicon nitride (5)by evaporation (6). The underlying silicon (1) is exposed by selectivelyremoving the overlying oxide and nitride materials covering one of themicrostructures using a combination of photolithography and etching (6).This structure will form a microchannel for actuating small volumes offluid.

The underlying silicon nitride within a second microstructure isselectively exposed by removing the overlying oxide layer only using acombination of photolithography and etching (8).

The exposed microstructures are permanently bonded to a carrier wafer(9) and the structure is inverted for further processing (10). The oxidematerial on the inverse side of the SOI wafer is selectively patternedusing a combination of photolithography and etching to expose the backside of each microstructure (11).

Example 16 Synthesis of Cleavable DNA-Biotin Construct

Synthesis of Non-Biotinylated Double-Stranded DNA (NP1): Twosingle-stranded 50-mers were synthesized using standard phosphoramiditechemistry (Integrated DNA Technologies). Oligo NP1-1S consisted of a 50nucleotide DNA sequence containing an amino group on the 5′-terminus,separated from the DNA by a C-12 carbon spacer (SEQ ID NO: 1) (1,MW=15,522.3 g/mole, ε=502,100 M⁻¹cm⁻¹). Oligo NP1-2AS consisted of a 50nucleotide DNA sequence complementary to NP1-1S (SEQ ID NO: 2) (2,MW=15,507.1 g/mole, ε=487,900 M⁻¹ cm⁻¹). Both oligonucleotides werequantitated and lyophilized prior to subsequent manipulation.

NP1-1S: 1 H₂N-AGTCATACGAGTCACAAGTCATCCTAAGATACCATACACATACCAA GTTCNP1-2AS: 2 GAACTTGGTATGTGTATGGTATCTTAGGATGACTTGTGACTCGTATGACTFinal ds-DNA Design- NP1: 3H₂N-AGTCATACGAGTCACAAGTCATCCTAAGATACCATACACATACCAAGTTCTCAGTATGCTCAGTGTTCAGTAGGATTCTATGGTATGTGTATGGTT CAAG

Synthesis of Non-Biotinylated Double-Stranded 50-bp DNA Construct:NP1-2AS (1.44 mg, 93.4 nmoles) was reconstituted in 0.5 mL distilledwater to give a 187 μM solution. NP1-1S (1.32 mg, 85.3 nmoles) wasreconstituted in 0.5 mL of 50 mM phosphate, 75 mM sodium chloride bufferpH 7.5 to give a 171 μM solution. The double-stranded construct (3) (SEQID NO: 1—forward strand (top); SEQ ID NO: 2—reverse strand (bottom)) wasmade by annealing 60 μL of NP1-1S solution (10.2 moles) with 40 μL ofNP1-2AS solution (7.47 moles). The mixture was placed in a heating blockat 85° C. for 30 min, followed by slow cooling to room temperature over2 hours. Double-stranded material was purified by injecting the totalannealing volume (100 μL) over a TosoH G3000SW column (7.8 mm×300 mm)equilibrated with 10 mM PBS buffer, pH 7.2. The column eluent wasmonitored at 260 and 280 nm. The double-stranded material (3) eluted at7.9 minutes (approx. 20 minutes). The DNA was concentrated to 150 μLusing a 0.5 mL Amicon filter concentrator (MW cut-off 10,000 Da). Thefinal DNA concentration was calculated to be 40.5 μM, as determined byA₂₆₀ absorbance.

Biotinylation of Single-Stranded 5′-Amino Oligo: A 100 mM solution ofsulfo-NHS-SS-Biotin (4, ThermoFisher Scientific) was made by dissolving6 mg of powder in 0.099 mL of anhydrous DMSO (Sigma Aldrich). Thesolution was vortexed and used immediately for biotinylating the5′-amino-DNA. Approx. 100 μL of ssDNA (1, 171 μM, 17.1 moles, 0.265 mg)(SEQ ID NO: 1) solution in 50 mM PBS, pH 7.5 was mixed with 3.4 μL of0.1 mM biotinylating reagent in DMSO (34.1 moles, 20-fold molar excessover the ssDNA). The mixture was mixed and allowed to react at roomtemperature for 2 hours. Two 0.5 mL Zeba spin desalting columns (MWcut-off 7,000 Da, ThermoFisher Scientific) were equilibrated in 10 mMPBS, pH 7.2. The crude biotinylated ssDNA solution was added to one Zebacolumn and eluted at 4,600 rpm for 1.3 minutes. The eluent wastransferred to a second Zeba column and eluted as described. Theconcentration of the purified NP1-1S—SS-Biotin (5) (SEQ ID NO:1 wasdetermined by measuring the A260 absorbance (2.03 mg/ml, 131 μM).

Formation of Biotinylated Double-Stranded DNA: Approximately 60 μL ofNP1-1S—SS-Biotin solution (5, 7.85 moles, 131 μM, 2.03 mg/mL) was mixedwith 42 μL of NP1-2AS solution (2, 7.85 moles, 187 μmon) (SEQ ID NO: 2).The solution was placed in a heating block at 85° C. for 30 minutes,followed by slow cooling to room temperature over 2 hours. Thedouble-stranded product was purified over a TosoH G3000SW column (7.8mm×300 mm) using 10 mM PBS, pH 7.2 by injecting the entire annealingvolume (approx. 100 μL). The double-stranded biotinylated materialeluted at 7.9 minutes (20 minutes run time), as monitored by A₂₆₀absorbance. The eluent volume was reduced to 480 μL using a 0.5 mLAmicon filter concentrator (MW cut-off 10,000 Da). The finalNP1-dithio-biotin (6) (SEQ ID NO:1—forward strand (top); SEQ IDNO:2—reverse strand (bottom)) concentration was calculated to be 16.3μM, as determined by A260 absorbance.

Example 17 Alternate Synthesis of Cleavable DNA-Biotin Construct

Complementary DNA Sequences (NP-31a and NP-31b): Two single-stranded60-mers were synthesized using standard phosphoramidite chemistry(Integrated DNA Technologies). Oligo NP-31a consisted of a 60 nucleotideDNA sequence containing an amino group on the 5′-terminus, separatedfrom the DNA by a C-6 carbon spacer (SEQ ID NO: 3) (1, MW=18,841.2g/mole, 1.7 μM/OD). Oligo NP-31b consisted of a 60 nucleotide DNAsequence complementary to NP-31a (SEQ ID NO: 4) (2, MW=18292.8 g/mole,1.8 μM/OD). Both oligonucleotides were quantitated and lyophilized priorto subsequent manipulation.

NP-31a: 1 H₂N-5′GCC CAG TGT CTT TGT AGG AGG AGC AGC GCG TCAATG TGG CTG ACG GAC CAT GGC AGA TAG3′ NP-31b: 25′CTA TCT GCC ATG GTC CGT CAG CCA CAT TGA CGC GCTGCT CCT CCT ACA AAG ACA CTG GGC3′ ds-DNA Desidn-NP-31: 3H₂N-5′GCC CAG TGT CTT TGT AGG AGG AGC AGC GCG TCAATG TGG CTG ACG GAC CAT GGC AGA TAG3′3′CGG GTC ACA GAA ACA TCC TCC TCG TCG CGC TGA TACACC GAC TGC CTG GTA CCG TCT ATC5′

Biotinylation of Single-Stranded 5′-Amino Oligo NP-31a: A 10 mM solutionof NHS-S-S-dPEG4-Biotin (4, MW=751.94 g/mole, Quanta BioDesign, Ltd) wasprepared by dissolving 15.04 mg of powder in 2.0 mL of dimethylformamide(Sigma Aldrich). The solution was vortexed and used immediately forbiotinylating the 5′-amino-DNA. Approx. 100 μL of ssDNA (1, 100 μM, 0.01μmoles, 0.188 mg) (SEQ ID NO: 3) solution in 10 mM phosphate bufferedsaline (PBS), pH 7.4 was mixed with 10 μL of 10 mM biotinylating reagentin DMF (0.1 moles, 10-fold molar excess versus the ssDNA). The mixturewas mixed and allowed to react at room temperature for 2 hours. Two 0.5mL Zeba spin desalting columns (MW cut-off 7,000 Da, ThermoFisherScientific) were equilibrated in 10 mM PBS, pH 7.2. The crudebiotinylated ssDNA solution was added to one Zeba column and eluted at4,600 rpm for 2 minutes. The eluent was transferred to a second Zebacolumn and eluted as described. The concentration of the purifiedNP-31-SS-Biotin (5) (SEQ ID NO: 3) was determined by measuring the A260absorbance (1.45 mg/mL, 77 μM).

Formation of Biotinylated Double-Stranded DNA: Approximately 60 μL ofNP-31-SS-Biotin solution (5, 77 μM) (SEQ ID NO: 3) was mixed with 50 μLof NP-31b solution (2, 100 μM). The solution was placed in a heatingblock at 85° C. for 30 minutes, followed by slow cooling to roomtemperature over 2 hours. The double-stranded product (SEQ IDNO:3—forward strand (top); SEQ ID NO:4—reverse strand (bottom)) waspurified over a TosoH G3000SW column (7.8 mm×300 mm) using 10 mM PBS, pH7.2 by injecting the entire annealing volume (approximately 100 μL). Thedouble-stranded biotinylated material eluted at 7.57 minutes asmonitored by A260 absorbance. The eluent volume was reduced using a 0.5mL Amicon filter concentrator (MW cut-off 10,000 Da). The finaldsNP-31-SS-Biotin (6) (SEQ ID NO: 3—forward strand (top); SEQ ID NO:4—reverse strand (bottom)) concentration was calculated to be 11 μM, asdetermined by A260 absorbance.

Example 18 Synthesis of Cleavable DNA-Biotin—Thiol-Mediated CleavageConstruct

Binding of ssNP-31-SS-Biotin to Streptavidin Coated MagneticMicroparticles (SA-MP) and Chemical Cleavage (TCEP or DTT): Chemicalcleavage experiments were performed on magnetic microparticles via thefollowing method (see FIG. 25). 100 μL of the modified oligonucleotidessNP-31-SS-Biotin solution at 77 μM in PBS, pH 7.2 was incubated with 1μL 0.1% Streptavidin paramagnetic microparticles for 30 minutes at roomtemperature. Excess oligo was removed by attracting the particles to amagnet and washing 10 times with PBST buffer, pH 7.4. The oligo-boundparticles were incubated with varying concentrations of either DTT orTCEP in PBS, pH 7.4 for 15 minutes. Microparticles were washed 10 timeswith PBST buffer, pH 7.4 to remove any cleaved oligonucleotide.Complementary sequence NP-31c (SEQ ID NO: 5) (7, MW=7494.6 g/mole, 5.2μM/OD) containing a fluorophore was incubated with the microparticlesfor 30 minutes in PBS, pH 7.4 to bind with any uncleavedssNP-31-SS-Biotin remaining intact on the particle. The microparticleswere attracted to a magnet and washed 10 times with PBST buffer, pH 7.4to remove any excess NP-31c segments. Coated microparticles prepared asabove that were washed but not subjected to chemical cleavage served asa control. The fluorescent signal on the particles was measured byfluorescence microscopy. Maximum cleavage efficiency was measured at 79%and 93% for DTT and TCEP, respectively as shown in Table 1.

NP-31c: 7 AlexaFluor ® 546-5′CTA TCT GCC ATG GTC CGT CAG3′

TABLE 1 Fluorescent Signal on Microparticles Cleavage (Relative LightUnits) Efficiency DTT (mM) 50 3579 79% 25 7417 57% 12.5 11642 32%0.78125 17052  0% 0 17059 (Control) TCEP (mM) 250 460 93% 222 448 94%187 474 93% 142 512 92% 83 477 93% 45 453 93% 19 452 93% 0 5023(Control)

Example 19 Synthesis of Cleavable DNA-Biotin—Photocleavage Construct

Evaluation of a Photocleavable DNA Sequence and Efficiency of Cleavageon Microparticles: A photocleavable sequence of single-stranded DNA wassynthesized using standard phosphoramidite chemistry (Integrated DNATechnologies). The oligonucleotide consisted of 48 nucleotides composingtwo Oligo segments (Oligo 8-1 (SEQ ID NO: 6) and Oligo 8-2 (SEQ ID NO:7)) separated by two photocleavable moieties (8, MW=15,430.1 g/mole,441800 L mol⁻¹ cm⁻¹). The 5′-terminus contained an amino group separatedfrom the DNA by a C-6 carbon spacer. A complementary strand to Oligo 8-2was synthesized containing a fluorescent tag (9, MW=7738.8 g/mole,212700 L mol⁻¹ cm⁻¹) (SEQ ID NO: 8). Both oligonucleotides werequantitated and lyophilized prior to subsequent manipulation.

Photocleavage experiments were performed on magnetic microparticles viathe following method (see FIGS. 26A and 26B). NP-8 was covalentlyattached to an antibody to generate an Ab-oligo complex (prepared byBiosynthesis Inc.). 100 μL of 33 nM antibody-oligo complex was incubatedwith 1 μL 0.1% solids of goat anti-mouse microparticles for 30 minutesat room temperature. Excess antibody-oligo complex was removed byattracting the particles to a magnet and washing 10 times with PBSTbuffer, pH 7.4. The microparticle complex solution was illuminated underUV light (300-350 nm wavelength) for 5 minutes. The microparticles wereattracted to a magnet and washed 10 times with PBST buffer, pH 7.4 toremove any cleaved Oligo segments. Following particle resuspension inPBST buffer, pH 7.4, fluorescently labeled Oligo 9 (SEQ ID NO: 8) wasadded to the irradiated microparticles and incubated for 30 minutes atroom temperature. Coated microparticles prepared as above that werewashed but not subjected to UV illumination (uncleaved Oligo 8-2) servedas a control. The fluorescent signal (AlexaFluor® 546) on the particleswas imaged by a fluorescence microscope. Cleavage efficiency when boundto paramagnetic microparticles was measured at 74% as shown in Table 2.

TABLE 2 Fluorescent Signal on Microparticles (Relative Light Units)Illumination 3660 No Illumination (Control) 13928 Cleavage Efficiency74%

Example 20 Thermal Cleavable Linkers

This Example describes thermal cleavable linkers and their cleavage.Such thermal cleavable linkers can be employed, for example, in a DMFchip, droplet-based microfluidic chip, SAW chip, or the like, asdescribed herein.

Thermal cleavable linkers are cleaved by elevating the temperature abovea threshold, such as in the thermal separation of double-stranded DNA.Temperature elevation in the DMF chip can be achieved photothermally bytransferring energy from light to an absorbing target. In one method, asource of light, such as a laser, having a wavelength of about 980 nm(range about 930 nm to about 1040 nm) can be applied to the DMF chip inthe region of the fluid sample. The light can be absorbed by the watermolecules in the fluid, resulting in an increase in temperature andcleavage of the linker. The level and duration of heating can becontrolled by pulse length, pulse energy, pulse number, and pulserepetition rate. For example, photothermal heating using the absorbanceband of water is described, e.g., U.S. Pat. No. 6,027,496.

Photothermal heating also can be achieved by coupling the light sourcewith a dye, or pigment containing target. In this case, a target area ofthe DMF chip is printed with an absorbing dye or pigment, e.g. carbonblack. When the fluid is in contact with the target, the light source,e.g. a commercially available laser diode, is directed at thelight-absorbing target, resulting in a localized increase in temperatureand cleavage of the linker. The level and duration of heating can becontrolled by the absorbance properties of the target, light wavelength,pulse length, pulse energy, pulse number, and pulse repetition rate. Forexample, photothermal heating using a light source in combination with alight absorbing target is described in U.S. Pat. No. 6,679,841.

In a third method of photothermal heating, an absorbing dye or pigmentcan be introduced into the fluid in the DMF chip. The light is thentransmitted through the DMF chip and the energy transferred to thedissolved or suspended absorbing material, resulting in a localizedincrease in temperature and cleavage of the linker. The level andduration of heating is controlled by absorbance properties of the targetmaterial, light wavelength, pulse length, pulse energy, pulse number,and pulse repetition rate. In one embodiment of this method, the lightabsorbing target is the magnetic microparticle suspension used in thedevice. For example, photothermal heating using suspended nanoparticlesin a fluid droplet is described in Walsh et al., Analyst, 140(5),1535-42 (2015). The reference of Walsh et al. also demonstrates some ofthe control that can be achieved in photothermal applications.

Example 21 Thermal Cleavage Accomplished Via Microwave-Induced ParticleHyperthermia

This Example describes the use of microwave-induced particlehyperthermia to facilitate thermal denaturation (such as dsDNAdenaturation, retro-Michael reactions, retro-Diels-Alder, and othereliminations) to release a countable moiety via a thermal sensitivecleavable linker, as immunoassay detection can be accelerated with theuse of low powered microwave radiation. Such thermal sensitive cleavablelinkers can be employed, for example, in a DMF chip, as describedherein.

In this example, formation of an orthogonally functionalized short dsDNAsegment such as a 15 bp sequence with a double stranded T_(m) in therange of 40-55° C. serves as the thermal release agent. The dsDNAsegment can be reacted with antibody via attachment chemistry such assulfhydryl-maleimide interaction and 26 nm carboxylated polystyrenenanoparticles (NP), such as those which can be obtained from Bangs Labs(Fishers, Ind., USA) via attachment chemistry such as amine-activatedcarboxylic acid chemistry. The 26 nm NPs have a surface charge of 528.7μeq/g and a parking area of 68.4 sq·Å/group (per manufacturerinformation). The antibody and nanoparticle are associated through thedsDNA segment which forms a thermally triggered releasable linker. Thethermal linker can be cleaved using a technique such as microwaveirradiation to trigger particle hyperthermia and a localized temperaturegradient.

DNA Sequence 1 (10) (SEQ ID NO: 9): H₂N-5′ CAA GCC CGG TCG TAA3′DNA Sequence 1b (11) (SEQ ID NO: 10): Maleimide-5′ TTA CGA CCG GGC TTG3′dsDNA Sequence (12) (SEQ ID NO: 9 - forward strand (top); SEQ ID NO: 10 - reverse strand (bottom)):H₂N-5′ CAA GCC CGG TCG TAA3′ 3′ GTT CGG GCC AGC ATT5′-Maleimide.

Annealing of the Orthogonally Functionalized Complementary DNA SequencesComplex: A solution of approximately 100 μM DNA Sequence 1 (SEQ ID NO:9) in PBS pH 7.5 can be mixed with 1.0 molar equivalents of DNA Sequence1b (theoretical T_(m) 51.6° C. per Integrated. DNA Technologies oligoanalyzer tool) (SEQ ID NO: 10) in PBS pH 7.5 and placed in a heatingblock at 60° C. for 30 minutes, followed by slow cooling to roomtemperature over 2 hours. The resulting dsDNA product is purified over aTosoH G3000SW column (7.8 mm×300 mm) using 10 mM PBS, pH 7.2 byinjecting the entire annealing volume. The eluent volume is reducedusing a 0.5 mL Amicon filter concentrator. The final dsDNA concentrationis determined by A260 absorbance. The reaction scheme is depicted below(“Mal” is maleimide).

Activation of carboxyl polystyrene nanoparticles and addition of doublestranded DNA: Carboxy nanoparticles are preactivated as described inExample 5 under section “Activation of carboxyl-polystyrenenanoparticles.” The DNA loading on the NP is determined by thermaldenaturation of the bound DNA strands, particle washing, annealing of afluorescently labelled complementary DNA sequence (such asAlexaFluor546-5′-TTA CGA CCG GGC TTG3′ (SEQ ID NO: 11)) and quantifiedusing a fluorescence microscope.

Antibody reduction and Conjugation to a NP-dsDNA Complex: The antibodyis reduced as described in Example 5 under section “Antibody reduction.”The reduced antibody can be used immediately for coupling to theNP-dsDNA complex. The resulting conjugate is centrifuged at 6,500 g andthe supernatant is removed via decanting. The wash procedure is repeated5 times with PBS pH 7.5 to remove any free antibody from thenanoparticle. The active antibody to nanoparticle incorporation ratiomay be quantified using a fluorescently labeled antigen to the givenantibody. The conjugate NP concentration (% solids) is determined usingUV-Vis spectroscopy (600 nm). The particle conjugate is examined by SEMand the size/charge distribution is determined using the ZetaSizer.

Microwave-induced Particle Hyperthermia and Nanopore CountingImmunoassay: The scheme above illustrates the nanopore counting assayutilizing the thermally denatured antibody-nanoparticle conjugate whosepreparation is described above. A sandwich type immunoassay can beprepared using magnetic microparticles coated with an analyte captureagent in which blood analyte is incubated with magnetic microparticles,washed, and incubated with the antibody-nanoparticle conjugatedescribed. Particle hyperthermia can be induced using microwaveirradiation to create a local temperature gradient near the particlesurface. Particle hyperthermia methods such as those reviewed in Dutzand Hergt (Nanotechnology, 25:452001 (2014)) may be used. The adaptationof these techniques to local thermal denaturation in an immunoassaysetting can provide a method to release a counting moiety (such as ananoparticle). Following removal of the magnetic microparticles, thecounting moiety (nanoparticle) is counted upon passage through thenanopore.

Example 22 Nanopore Counting Data

This Example describes nanopore counting data for a variety of tags,e.g., ssDNA hybrid molecules with polyethyleneglycols (DNA-STAR), dsDNA,dsDNA labeled with DBCO, and PAMAM succinamic acid dendrimers. Use ofthese different tags along with different size nanopores was done toprovide for nanopore optimization. Different molecular polymer labelswere suspended in an appropriate salt buffer and detected using astandard fluidic cell cassette.

Current-voltage (i-V) recordings (voltammetric data) and current-time(i-t) recordings were recorded using in-house instrumentation. Acomputer software program called CUSUM was employed to run through theacquired data and detect events based on the threshold input by theuser. Any impact of subjectivity in the assessment was minimized bydetection of as many events as possible and filtering afterwards forspecific purposes.

Initial experiments were performed with the tags added to the cis sideof the membrane. An electric bias of 200 mV was applied to the labelsolution and current blockades were monitored using the Axopatch 200Bamplifier and CUSUM software.

It is known that small molecules can go through nanopores quite fastunless the pore size restricts their passage. The current blockages offast events can be deformed due to the limited bandwidth of a system.Faster molecules can even be completely undetected by a particularsystem.

In our studies, only larger polymers and molecules labeled with largegroup modifiers were detected. Experimental conditions and number ofdetectable events are shown in Table 3

TABLE 3 Electrolyte Membrane Nanopore Detection Events Cconc BackgroundConcentration Thickness Diameter Voltage detected by Polymer (nM)Electrolyte (M) pH (nm) (nm) (mV) CUSUM 50 bp dsDNA 60 LiCl 3.6 8.0 103.9 200 414 control DBCO 96 LiCl 3.6 8.0 10 3.9 200 594 backbone dsDNastar 20 LiCl 3.6 8.0 10 3.9 200 5589  PAMAM 100  KCl 1.0 10 10 7.8 100264 (6^(th) gen) - 150 1122  succinamic acid 200 1322 

These data confirm that DNA dendrimers, polymers, and PAMAM dendrimerscan be used as detection labels for solid-state nanopore sensors.

Example 23 Nanopore Differentiation of Biomolecules

In this Example, the nanopore was used to differentiate biomolecules(e.g., dsDNA stars, DBCO-modified dsDNA and regular dsDNA). Thismethodology can be used for multiplexing using different label types.

This Example employed a 50 bp oligonucleotide containing a branch pointin the middle (bp #25), where a single-stranded oligonucleotide wascovalently linked (DNA-Star); a double-stranded 50 bp oligonucleotidecontaining a dibenzylcyclooctyne (DBCO) modification in the middle (base#25); and a 5′-thiol modified double-stranded DNA oligonucleotide.

These various modified DNA molecules were analyzed using three differentSiN_(x) nanopores in 3.6 M LiCl buffer. DNA-star molecules were analyzedwith a 4.0 nm diameter pore; DBCO-modified DNA was analyzed with a 3.7nm diameter pore; thiol-modified DNA was analyzed with a 4.2 nm diameterpore. Current blockade levels (pA) were plotted against nanoporeduration times (μsec), in order to show the ability of the nanopores todifferentiate the three different biomolecules. At a population level,the three different labels appear to be distinguishable, as demonstratedby the distinct pattern differences in the scatter plots (FIGS.24A-24C). Identification of individual events in real-time requiresadditional levels of blockade level and time information as a way todistinguish signals from noise. The ability to differentiate differentnanopore labels demonstrate that nanopores can be employed formultiplexing in various assays.

Example 24 Qualitative Analysis

The following example describes a method for conducting a qualitativeassay. Basically, in this example, a construct was used to demonstratethe principle of the assay on a DMF chip and the construct was cleavedand the label was released and then counted using a nanopore so as togenerate a signal as it translocates the nanopore, thus indicating thatthe binding of two specific binding member pairs (streptavidin andbiotin) wherein this cleavage and subsequent counting of a dsDNA labelis correlated to the specific binding having occurred during the assay.Furthermore, appropriate control experiments were conducted to confirmthat the signal generated from the label that was counted during thenanopore translocation measurement was due to the specific binding eventhaving occurred during the assay process rather than being correlated tothe presence of thiol cleavage reagent being introduced into the assayprocess flow. The details of the experiments conducted follow.

Thiol-Mediated Cleavage Using DMF: A biotin-labeled double-stranded DNAcontaining a cleavable disulfide bond ((6) of Example 17) was used as atarget for nanopore detection/counting. The binding assay consisted ofbinding the biotin DNA to streptavidin magnetic microparticles on a DMFchip, followed by a thiol-mediated chemical cleavage step (see FIG. 25).Reagent placement on the DMF chip is shown in FIG. 27. The cleaved DNAtarget, separated from the species bound to the streptavidin magnetparticles, was transferred to a nanopore fluidic cell containing asolid-state silicon nitride (SiN_(x)) membrane with a pre-drillednanopore created by controlled dielectric breakdown (H. Kwok, et al.,PLoS, 9(3), 2014). The DNA target material was counted and analyzedusing open-source CUSUM software analysis package (NIST).

Appropriate reagents were loaded onto a glass DMF chip (3″×2″×0.0276″)containing 8 reagent reservoirs. Except for waste reservoirs, eachreservoir contained approx. 5 μL of each reagent. Concentrations ofreagents were as follows: 11 μM Biotin-SS-DNA in PBS (pH=7.2); 10 mg/mL(w/v) M-270 2.7 μm streptavidin-coated magnetic microparticles (LifeTechnologies); PBS wash buffer (pH=7.2)+0.05% ETKT (Ethylene tetra-KIS(ethoxylate-block-propoxylate) tetro), 50 mMtris-(2-carboxyethyl)phosphine (TCEP). Approximate size of a dispensedDMF droplet was 1.5

One droplet of M-270 streptavidin-coated microparticles was dispensedand mixed with 1 droplet of dsNP-31-SS-biotin for approx. 40 minutes.Mixing was accomplished by combining the 2 droplets and moved in acircular pattern on the DMF chip over 12 electrodes (3×4). The bottommagnet was engaged to collect the microparticles and the supernatant wasmoved to a waste reservoir. Next, two droplets of PBS/ETKT buffer weredispensed and moved to the microparticle slug, which was thenresuspended in solution. The suspension was mixed for 5 minutes beforethe magnet was again engaged and the supernatant was removed to thewaste reservoir. The particle wash step was repeated a total of 11times, while gradually increasing the mixing time up to 45 minutes. Thelast wash supernatant was moved to an empty reservoir. An additional 5droplets of PBS/ETKT was moved to the same reservoir. The wash andPBS/ETKT in the reservoir was removed using a 34-AWG nonmetallic syringe(Microfil 34-AWG) and transferred to a 1.5 mL Eppendorf tube, inpreparation for nanopore analysis. Cleavage was initiated by moving 2droplets of TCEP reagent to the microparticle slug and mixing for 45minutes. The bottom magnet was engaged and the supernatant (containingthe cleaved DNA) was moved to an empty reservoir. An additional 5droplets of PBS/ETKT wash buffer was moved to the same reservoir. Thefinal extract was removed from the DMF chip using the 34 gaugenonmetallic syringe and transferred to a 1.5 mL Eppendorf tube, inpreparation for nanopore analysis. The cleavage eluent was microfugedfor 30 seconds and placed in a magnetic rack for 1 minute, to remove anytrace microparticles.

Nanopore Analysis: Nanopore fabrication was achieved using controlleddielectric breakdown (CBD) of a 10 nm thick SiN_(x) membrane embedded ina TEM window (0.05 μm×0.05 μm) (Norcada NT0052, low stress SiN_(x)).This method is capable of producing small diameter solid-state poreswith high precision and minimal cost. The TEM-SiN_(x) membrane wasplaced in a polytetrafluoroethylene (PTFE) fluidic cell containing twobuffer chambers, and sealed using two silicone elastomer gaskets. Thefluidic cell contained a 16 μL volume channel in the bottom of the cell,which connected the salt solution in the upper chamber to the nanoporemembrane. For nanopore fabrication, the fluidic cell was first filledwith degassed ethanol, exchanged with degassed deionized water and thenfilled with degassed 0.5 M KCl, buffered to pH 10 with sodiumbicarbonate in 18 MΩ deionized water. Fabrication was performed using anamplifier using a bias voltage of 8V. The two sides of the fluid cellwere connected using silver/silver chloride wires. As described in Kwoket al, while setting a fixed voltage of 8V, the current exhibits acapacitance (reduction of current) in real time. When the currentincreases, the power is removed from the cell. The sampling rate for thefabrication=25 KHz. An increase of the leakage current indicatesformation of a pore, whereby the voltage was turned off. The porediameter was determined from the following conductance-based equation:

$G = {\sigma\left( {\frac{4L}{\pi\; d^{2}} + \frac{1}{d}} \right)}^{- 1}$

where G=conductance, σ=bulk conductivity (12.35 S/m measured for KCl),L=thickness of the membrane (10 nm), d=pore diameter (S. Kowalczyk, A.Grosberg, Y. Rabin, C. Dekker, Nanotech., 22, 2011). The nanopore waschecked for ohmic behavior by generating an I-V curve. The measureddiameter of the nanopore was determined to be 4.4 nm, and wassubsequently used for detection of the cleaved ds-SS-DNA target.

The fabrication salt buffer was replaced with 3.6 M LiCl, which was usedas the sensing buffer for detecting translocation events. A headstagewas placed between an Axopatch 200B amplifier and the silver/silverchloride connection to the fluidic cell housing the nanopore membrane.

Approx. 0.2 μL of the TCEP-cleaved ds-DNA target was diluted with 1.8 μLPBS buffer (this represented a 10-fold dilution of the TCEP-cleavageeluent), and the entire volume was loaded and mixed into the nanoporecell chamber, which contained approximately 30 μL of 3.6 M LiCl saltsolution. The last DMF wash eluent was used as a negative cleavagecontrol (this was not diluted). The number of DNA translocations wasmeasured for 23 and 65 minutes for the TCEP eluent and negative control,respectively and converted to a flux rate (sec⁻¹). The results depictedin FIG. 28 demonstrate that the ds-SS-DNA target was successfullycleaved from the M-270 streptavidin particles using DMF and detectedusing a solid-state nanopore as a detector. SNR was determined to be21.9, as measured from the nanopore flux rate.

Data Analysis: The number of translocation events were determined byfirst calculating the anticipated current change found in a doublestranded DNA translocation event under experimental test conditionsusing the equation

$\begin{matrix}{{{\Delta G} = \frac{\sigma\pi d_{DNA}^{2}}{4L}},} & ({S1})\end{matrix}$

as referenced in Kwok et al., “Nanopore Fabrication by controlledDielectric Breakdown” Supplementary Information Section 8 and Kwok, H.;Briggs. K.; and Tabard-Cossa. V.; “Nanopore Fabrication by ControlledDielectric Breakdown”—PLoS ONE 9(3): e92880 (2014). Using thisanticipated current blockage value, the binary file data of theexperimental nanopore output was visually manually scanned foracceptable anticipated current blockage events. Using these events, theThreshold and Hysteresis parameters required for the CUSUM nanoporesoftware were applied and executed. The output from this software wasfurther analyzed using the cusumtools readevents.py software andfiltering blockage events greater than 1000 pA (as determined from thefirst calculation). The flux events, time between events and othercalculations were determined from the readevents.py analysis tool.Additional calculations were made on the CUSUM generated data using JMPsoftware (SAS Institute, Cary, N.C.). It is understood that this methodof threshold setting is one approach to data analysis and setting athreshold and that the present invention is not limited to this methodand that other such methods as known to those skilled in the art canalso be used.

Summary: This example describes a qualitative assay by conducting theprocess of steps as described herein. A direct assay was conducted usingthe cleavable linker conjugate, as described in Example 17, with a thiolbased cleavage step, as shown in FIG. 25. It is understood that othercleavable linker approaches to conducting such an assay may alsoinclude, but are not limited to, various other methods of cleavage of alinker so as to allow for the counting of various tags, as describedherein. For example, such other alternative cleavage methods and/orreagents in addition to the method described in Example 17 can includethose described in Example 16, Example 18, Example 19, Example 20 andExample 21, in addition to other cleavage methods described herein andknown to those skilled in the art. It is also understood that while theassay format demonstrated in this Example (Example 24) represents adirect assay, other formats such as sandwich immunoassay formats and/orvarious competitive assay formats, such as are known to those skilled inthe art, can be implemented as well to conduct an assay using thedescribed methods.

For example, the sandwich immunoassay format for the detection of TSH(thyroid stimulating hormone), as described in Example 9, demonstratedthe ability to conduct such an assay on a low cost DMF chip.Additionally, a number of various bioconjugation reagents useful for thegeneration of immunoconjugate or other active specific binding membershaving cleavable linkers can be synthesized using variousheterobifunctional cleavable linkers such as those described in Example1, Example 2, Example 3, Example 4, Example 5 and Example 6, in additionto other cleavable linkers that are otherwise known to those skilled inthe art. Immunoconjugates useful for the practice of the presentinvention can be synthesized by methods such as those described inExample 3, Example 4, Example 5 and Example 6 as well as by methodsknown to those skilled in the art. Additionally, Example 8 shows thefunctionality of various fluidic droplet manipulations on a low costchip that can facilitate various steps needed to carry out various assayformats including sandwich and competitive assay formats as well asother variations thereof known to those skilled in the art. Example 11shows the fabrication of a nanopore that can be used to count cleavablelabel in an assay but it is understood that other methods for nanoporefabrication known to those skilled in the art can also be used for thispurpose. Example 16 also represents another construct useful for theconduct of an assay where a cleavage is effected, thus leading to acountable label being released so as to be countable using the nanoporecounting method, as described within this example.

Example 22 shows generally how counting can be effected so as to be ableto measure translocation events relating to the presence of a variety oflabels traversing the nanopore. FIG. 29 shows the concept ofthresholding of the signal so as to be able to manipulate the quality ofdata in a counting assay. FIG. 28 shows qualitative assay data that isrepresentative of the type of data that can be used to determine thepresence of an analyte using such assay methods as described within thisexample. It is also understood that while dsDNA was used as a label inthis particular example, other labels, such as the label described inExample 5 and/or Example 22 can also be utilized, including, but notlimited to nanobeads, dendrimers and the like. Such constructs as neededto generate appropriate reagents can be synthesized through variousexamples herein this application or otherwise via methods known to thoseskilled in the art.

Example 25 Quantitative Analysis

The following example describes a method for conducting a quantitativeassay. Basically, in this example, and as an extension of Example 24, astandard curve was generated so as to demonstrate that increased amountsof counting label, in this case with the countable label being a dsDNAmolecule, correlated on a standard curve to the amount of specificbinding agent that has been bound (which it turn correlates to theamount of analyte existing in the original sample) in an assay (binding)step. The standard curve for this particular experiment can be found inFIG. 31, FIG. 32, FIG. 34 based on various different methods of dataanalysis or FIG. 34 which relies up flux to generate a standard curve.In the latter case, the measurement method shown in FIG. 34 based uponthe events/time (flux of counting events) but it is understood thatother measurement methods can also be used to generate a standard curvecorrelating to the amount of analyte concentration being measured in agiven sample. The details of the experiments conducted are as follows.

Nanopore Fabrication: Nanopore fabrication was achieved using controlleddielectric breakdown (CBD) of a 10 nm thick SiN_(x) membrane embedded ina TEM window (0.05 μm×0.05 μm) (Norcada NT0052, low stress SiN_(x)) asthis method is capable of producing small diameter solid-state poreswith high precision and minimal cost. The TEM-SiN_(x) membrane wasplaced in a polytetrafluoroethylene (PTFE) fluidic cell containing twobuffer chambers, and sealed using two silicone elastomer gaskets. Thefluidic cell contained a 16 μl volume channel in the bottom of the cell,which connected the salt solution in the upper chamber to the nanoporemembrane. For nanopore fabrication, the fluidic cell was first filledwith degassed ethanol, exchanged with degassed deionized water and thenfilled with degassed 0.5 M KCl, buffered to pH 10 with sodiumbicarbonate in 18 MΩ deionized water. Fabrication was performed using anamplifier using a bias voltage of 8V. The two sides of the fluid cellwere connected using silver/silver chloride wires. As described in Kwoket al, while setting a fixed voltage of 8V, the current exhibits acapacitance (reduction of current) in real time. When the currentincreases, the power is removed from the cell. The sampling rate for thefabrication was 25 KHz. An abrupt increase of the leakage currentindicated formation of a pore, whereby the voltage was turned off. The0.5 M KCl buffer was replaced with 3.6 M LiCl (pH=8.3).

The pore diameter was determined from the following conductance-basedequation:

${G = {\sigma\left( {\frac{4L}{\pi\; d^{2}} + \frac{1}{d}} \right)}^{- 1}},$

where G=conductance, σ=bulk conductivity (16.06 S/m measured for LiCl),L=thickness of the membrane (10 nm), and d=pore diameter (S. Kowalczyk,A. Grosberg, Y. Rabin, C. Dekker, Nanotech., 22, 2011). The nanopore waschecked for ohmic behavior by generating an I-V curve. The measureddiameter of the nanopore was determined to be 4.8 nm, and wassubsequently used for detection of the DNA calibration standards.

DNA Dose-Response: DNA standards were used as calibrators to observe adose-response curve by determining the change in nanopore flux rate withincreasing concentrations of DNA. This generated a standard curve, whichcan be used for quantitation of a cleaved DNA label in an immunoassay.Two μl of a 1.5 μM 100 bp DNA standard (ThermoScientific) was pipettedinto the PTFE fluidic cell containing 30 μl of 3.6 M LiCl salt solution,to give a final concentration of 94 nM DNA. The reagent was mixed bypipetting the solution up and down several times prior to nanoporeanalysis. The cell was subjected to a DC bias of +200 mV and monitoredfor current blockades over 60 minutes. CUSUM analysis software was usedto characterize electrical signals and count rates. This procedure wasrepeated two times to give two additional points on the standard curve,182 nM and 266 nM. Current blockades over different time periods areshown for all three standards—41 seconds for 94 nM (FIG. 30A); 24seconds for 182 nM (FIG. 30B); 8 seconds for 266 nM (FIG. 30C). Baselinenoise was empirically estimated to be approximately 900 pA, 900 pA and1,000 pA for FIG. 30A, FIG. 30B and FIG. 30C, respectively.

Data from the run was used to generate three different types ofdose-response curves—number of events over a fixed amount of time (5minutes) (FIG. 31); time required for fixed number of events (200events) (FIG. 32); and events per unit time (FIG. 33). Each of thesecurves may be used as a standard curve for a quantitative nanopore-basedimmunoassay, using DNA as the label. Similarly, other labels may be usedto quantitate various analytes, such as dendrimers, polymers,nanoparticles, and the like.

Seq31-SS-Biotin DNA Dose-Response: The synthetic DNA construct,Seq31-SS-biotin, was used as the source material to generate adose-response curve (FIG. 47). This target can be used to quantitate thecleaved label NP-Seq31-SS-biotin, which was cleaved from thestreptavidin beads in the qualitative assay. Since this material hasapproximately the same MW and charge density as the cleaved labelSeq31-SS-biotin, it may be used in a calibration curve to quantitate thecleaved target from streptavidin microparticles using TCEP and/or DTT.

Data Analysis: The number of translocation events were determined byfirst calculating the anticipated current change found in a doublestranded DNA translocation event under experimental test conditionsusing the equation:

$\begin{matrix}{{\Delta G} = \frac{\sigma\pi d_{DNA}^{2}}{4L}} & ({S1})\end{matrix}$

as referenced in Kwok et al., “Nanopore Fabrication by controlledDielectric Breakdown” Supplementary Information Section 8 and Kwok, H.;Briggs, K.; and Tabard-Cossa, V.; “Nanopore Fabrication by ControlledDielectric Breakdown”—PLoS ONE 9(3): e92880 (2014). Using thisanticipated current blockage value, the binary file data of theexperimental nanopore output was visually manually scanned foracceptable anticipated current blockage events. Using these events, theThreshold and Hysteresis parameters required for the CUSUM nanoporesoftware were applied and executed. The output from this software wasfurther analyzed using the cusumtools readevents.py software andfiltering blockage events greater than 1000 pA (as determined from thefirst calculation). The flux events, time between events and othercalculations were determined from the readevents.py analysis tool.Additional calculations were made on the CUSUM generated data using JMPsoftware (SAS Institute, Cary, N.C.). It is understood that this methodof threshold setting is one approach to data analysis and that thepresent invention is not limited to this method but other such methodsas known to those skilled in the art can also be used.

Summary: This example describes a quantitative assay by conducting theprocess of steps as described herein. A direct assay was conducted usingthe cleavable linker conjugate, as described in Example 17, with a thiolbased cleavage step, and as shown in FIG. 25. It is understood thatother cleavable linker approaches to conducting such an assay may alsoinclude, but are not limited to, various other methods of cleavage of alinker so as to allow for counting of various tags using a nanopore, asdescribed herein. For example, such other cleavage methods in additionto the method described in Example 17 can include, but is not limitedto, those described in Example 18, Example 19, Example 20 and Example21, in addition to other methods described herein and known to thoseskilled in the art. It is also understood that while the assay formatdemonstrated in this Example (Example 25) represents a direct assay,other formats such as sandwich immunoassay formats and/or variouscompetitive assay formats, such as are known to those skilled in theart, can be implemented as well to conduct an assay.

For example, the sandwich immunoassay format for the detection of TSH(thyroid stimulating hormone), as described in Example 9, demonstratedthe ability to conduct such an assay on a low cost DMF chip.Additionally, a number of various bioconjugation reagents useful for thegeneration of immunoconjugate or other active specific binding membershaving cleavable linkers can be synthesized by those skilled in the artusing various heterobifunctional cleavable linkers and conjugatessynthesized by methods such as those described in Example 1, Example 2,Example 3, Example 4, Example 5 and Example 6, in addition to othercleavable linkers or conjugates that could be synthesized by methodsthat are known to those skilled in the art. Additionally, Example 8shows the functionality of various fluidic droplet manipulations on alow cost chip that can facilitate various steps needed to carry outvarious assay formats including sandwich and competitive assay formatsas well as other variations thereof known to those skilled in the art.Example 16 also represents another construct useful for the conduct ofan assay where a cleavage is effected, thus leading to a countable labelbeing released so as to be countable using the nanopore counting methodas described within this example.

Example 22 shows generally how counting can be performed so as to beable to measure translocation events relating to the presence of a labeltraversing the nanopore. FIG. 29 shows the concept of thresholding ofthe signal so as to be able to manipulate the quality of data in acounting assay. FIGS. 31, 32 and 33 show quantitative assay data outputthat is representative of the type of data that can be used to determinethe amount of an analyte using such assay methods as described withinthis example. FIG. 34 shows a standard curve generated from a constructthat has been cleaved using a chemical method. It is also understoodthat while dsDNA was used as a label in this particular example, otherlabels, such as the label described in Example 5, can also be utilized,including, but not limited to, nanobeads, dendrimers and the like. Suchconstructs can be synthesized via methods known to those skilled in theart.

Example 26 Nanopore Electrical Field Simulations

A series of COMSOL simulation runs were performed on the proposednanopore membrane design used in the silicon module, to study theinfluence of the size of the SiO₂ via on the counter ion concentrationand electroosmotic flow rate through a theoretical 10 nm diameternanopore. A top layer of SiO₂ served multiple purposes—1) provide aninsulating layer to the SiN_(x) membrane and, thereby, reduce thecapacitive noise of the nanopore; 2) to increase the robustness andstrength of the SiN_(x) membrane within the silicon substrate; 3) todecrease the size of the SiN_(x) area exposed to solution, therebyimproving positioning of the pore on the membrane from the controlleddielectric breakdown (CBD) process. Electrical field simulations wereused to determine interference of the SiO₂ layer on localized counterion concentration and electroosmotic flow through the pore.

With reference to FIG. 35, the silicon substrate (1) was etched to givecis and trans chambers, situated above and below the SiN_(x) membrane.The SiN_(x) membrane (50 μm×50 μm) (2) was layered between a 300 μmthick bottom layer of SiO₂ and a 300 μm thick top layer of SiO₂ (3). Thetop layer was fabricated to form a SiO₂ via (4), which allowed formationof the nanopore during CBD. The optimal diameter of the SiO₂ via wasdetermined by the simulation.

COMSOL Simulation Results: COMSOL electrical field simulations usedphysical models based on materials, electrostatics, molecular transportand Laminar flow properties. Electric potential was based on Poissonequation; ionic flux was based on Nernst-Planck equation; fluid velocitywas based on Stokes equation. Physical parameters used for thesimulation are defined in Table 1, shown in FIG. 36.

COMSOL results for counter ion concentration gradients near the pore areshown in FIG. 37, and show little to no influence of the ionicconcentration when the SiO₂ via diameter was >50 nm in diameter. Below50 nm, an accumulation of net charge near the mouth of the poreresulted. The most severe effect was observed at a diameter of 25 nm,where a large ionic gradient formed near the pore. The results showed afairly large influence of the SiO₂ surface when the nanopore was lessthan 25-50 nm away from the SiO₂ wall.

Electroosmotic flow rates of counter ions through the pore weresimulated as a way to determine any influence the SiO₂ layer may have onnanopore sensing (FIG. 38). The highest rate of electroosmotic flowoccurred with the larger via diameters (100-4,500 nm). A reduction inflow rate through the pore was observed for a 50 nm SiO₂ via, followedby a significant reduction for a 25 nm via.

As shown in FIG. 39, measurement of conductance through the pore vs. viadiameters showed a saturation curve above 100 nm, with diminishingconductance as the via diameter was reduced in size from 100 nm to 25nm.

Example 27 Integrating a Nanopore Module into a Digital Microfluidic(DMF) Module

The nanopore module was located on one side of the DMF module. A holewas present in the DMF module to allow liquid transport from the DMFmodule to the nanopore module for pore creation and analyte detection(e.g., see FIG. 40).

One electrode from the nanopore module terminated within the fluidvolume in the nanopore module. The other electrode terminated within thefluid volume in the DMF module. This electrode was routed through asecond hole in the DMF module. To demonstrate that liquid was able tomove through the hole within the DMF module, a flat piece of paper waspushed over the exterior surface of the chip after liquid was moved inplace. The wetting of this paper showed that the liquid was able to movefrom the DMF module to another module located above this hole viacapillary forces (FIG. 41).

With reference to FIG. 42, the DMF module was equipped with Ag/AgClelectrodes for control of the nanopore fabrication. In this setup, theliquid volume on the nanopore module was an open-air droplet of LiCl.This liquid was dispensed directly onto the nanopore module and theelectrode terminal was suspended within this droplet.

The sample was moved to the hole in the DMF module using DMF technology.The sample passively migrated through the hole to become exposed to thenanopore module for nanopore creation. The nanopore module is sealed tothe DMF module (e.g. using PDMS, pressure, wax, etc.), isolating theliquid volumes held within each module. FIG. 45 shows the current as afunction of time during the fabrication of the nanopore.

Once the nanopore was created, a conditioning process (varying voltageover time) was used to physically modify the nanopore and clean thesignal. This process improved symmetry in the I-V curve. The before andafter I-V curves are shown in FIGS. 46A and 46B, respectively.

Example 28 Counting Labels and Pore Size Analysis

A set of experiments were run using double stranded DNA under varioussets of conditions to analyze and demonstrate certain attributesrelative to pore size and counting label size. In these experiments,various parameters were explored including detection voltage, DNAlength, DNA concentration, salt concentration and salt composition,membrane material, membrane thickness, nanopore diameter and otherfactors.

The data set was then analyzed relative to signal to noise ratio andcompared that factor to various pore size relative to counting labelsize (estimated molecular diameter). Certain factors such as membranematerial and thickness, for example, were held constant in this set ofexperiments, while other factors were varied.

From an aggregate data set analysis, the averages of ratios were plottedbetween counting label average diameter and nanopore size to the SNR(signal to noise ratio) determined in the experiment (FIG. 47). FIG. 47demonstrates generally that useful counting data can be obtained from arange of such ratios, in this particular data set from between around0.4 to 0.8 in such ratio—assuming a molecular diameter of a dsDNA ofaround 2.0 nm approximately. Linear dsDNA is known from the literatureto be about that molecular diameter and the analysis assumes the DNAthreads through the pore in its linear conformation. Table 4 shows thecalculated data.

TABLE 4 AVERAGE PORE LABEL MOLECULAR DIAMETER TO PORE RATIO SNR 0.64527.5 0.556 53.7 0.476 12 0.714 23.7 0.803 17 0.645 22.5 0.8 20.2 0.58817 0.69 55 0.476 23.5

While conditions varied, as previously mentioned in this example, thegeneral range in this data set shows that counting data with reasonablesignal to noise can be obtained within this range. Furthermore, itshould be noted that one skilled in the art would recognize that othercounting label molecular diameter to nanopore diameter ratios could beutilized to achieve reasonable SNR. Additionally, it would be recognizedby one skilled in the art that generally a label should have at leastone dimension of its molecular diameter that is less than the size ofthe nanopore so as to be able to pass through the pore, or in otherwords, this ratio of label molecular diameter to nanopore diametershould generally be less than one for the label to be able to passthrough the pore, except in cases perhaps where conditions such as aredescribed in a technology called nanopore force spectroscopy is used,wherein energy is added to the system to facilitate conformationalchanges to occur in the label and thus allow it to pass through the poreafter deformation to a level that would allow such a translocation eventto occur.

It should also be understood to one skilled in the art that other labelscan be utilized for counting other than dsDNA as described in thisexample, and that they may have different behaviors than that shown inthis graph. Furthermore, it should also be understood that it ispossible to also obtain acceptable SNR from other molecular diameter tonanopore ratios to enable molecular counting of such labels, and thatcurrent blockage can be related to molecular diameter of such a countinglabel as described in the equation below

$\begin{matrix}{{\Delta G} = \frac{\sigma\pi d_{DNA}^{2}}{4L}} & ({S1})\end{matrix}$

which can be found in the following references: Kwok et al., “NanoporeFabrication by controlled Dielectric Breakdown” SupplementaryInformation Section 8 and/or Kwok, H.; Briggs, K.; and Tabard-Cossa, V.;“Nanopore Fabrication by Controlled Dielectric Breakdown”—PLoS ONE 9(3):e92880 (2014). This equation can be used in order to gate or thresholdsignal as described in Examples 24 and 25 in this document.

Certain specific conditions varied within this aggregate set of nanoporecounting experiments included:

-   -   Ionic Strength—either 3 or 3.6 M    -   DNA length—10 kbp, 50 bp or 1 kbp    -   Ionic Salt Used—either LiCl or KCl    -   Membrane Material—SiN_(x) (constant throughout data set)    -   Membrane Thickness—10 nm (constant throughout data set)    -   DNA Concentration(s)—varied between 3 nM and around 306 nM    -   Voltages—varied including increments between 50 and 600 mV    -   Nanopore Diameter—a variety of pore sizes including 8.0, 1.1,        3.6, 4.2, 2.8, 2.5, 7.7, 3.1, 2.7, 2.6, 2.9 and 4.2 (all in        nanometers).

Conclusions can be drawn that various conditions, including but notlimited to these, show that one can obtain in situations where thecountable label is smaller than the diameter of the pore can cause ablockage of the flux of ion current across the pore when a voltage isapplied as per the amount as calculable but this equation of Kwok et alas referenced in this example [Kwok et al., “Nanopore Fabrication bycontrolled Dielectric Breakdown” Supplementary Information Section 8and/or Kwok. H.; Briggs, K.; and Tabard-Cossa, V.; “Nanopore Fabricationby Controlled Dielectric Breakdown”—PLoS ONE 9(3): e92880 (2014)].

It is also understood that these conditions can be applied to showcounting label molecular diameters to pore diameters that will functionwith reasonable signal to noise for other labels besides dsDNA,including but not limited to dendrimers, hemi-dendrimers, nanobeads,anionic or cationic polymers, denatured linearized aptamers, negativelyor positively charged poly peptides or other charged polymers orcountable molecular entities and the like.

Finally, although the various aspects and features of the invention havebeen described with respect to various embodiments and specific examplesherein, all of which may be made or carried out conventionally, it willbe understood that the invention is entitled to protection within thefull scope of the appended claims.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the invention are setout in the following numbered clause:

Clause 1. A method for measuring or detecting an analyte present in abiological sample, the method comprising: (a) contacting the sample witha first binding member, wherein the first binding member is immobilizedon a solid support and wherein the first binding member specificallybinds to the analyte; (b) contacting the analyte with a second bindingmember, wherein the second binding member specifically binds to theanalyte and wherein the second binding member comprises a cleavable tagattached thereto; (c) removing second binding member not bound to theanalyte bound to the first binding member; (d) cleaving the tag attachedto the second binding member bound to the analyte bound to the firstbinding member; (e) translocating the tag through one or more nanoporesin a layer; and (f) assessing the tag translocating through the layer,wherein measuring the number of tags translocating through the layermeasures the amount of analyte present in the sample, or whereindetecting tags translocating through the layer detects that the analyteis present in the sample.

Clause 2. The method of clause 1, wherein each tag translocating throughthe layer is a translocation event and measuring the number oftranslocation events measures the amount of analyte present in thesample, wherein the amount of analyte present in the sample isdetermined by: i) counting the number of translocation events during aset period of time and correlating the number of translocation events toa control; ii) measuring the amount of time for a set number oftranslocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference standardcomprising a calibration curve, standard addition, or digital polymerasechain reaction.

Clause 3. The method of clauses 2, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

Clause 4 The method of any one of clauses 1 to 3, wherein the methodinvolves single molecule counting.

Clause 5. The method of any one of clauses 1 to 4, wherein the tag isselected from the group consisting of an anionic polymer, a cationicpolymer, a dendrimer, and a nanoparticle.

Clause 6. The method of any one of clauses 1 or 5, wherein the tag issubstantially spherical or hemi-spherical.

Clause 7. The method of any one of clauses 1 to 6, wherein the tag issubstantially spherical and comprises a nanoparticle.

Clause 8. The method of any one of clauses 1 to 7, wherein the tag issubstantially spherical or hemi-spherical and comprises a dendrimer.

Clause 9. The method of clauses 8, wherein the dendrimer is positivelyor negatively charged.

Clause 10. The method of clauses 5 or 7, wherein the nanoparticlecomprises a positively charged nanoparticle.

Clause 11. The method of clauses 10, wherein the nanoparticle comprisesa negatively charged nanoparticle.

Clause 12 The method of any one of clauses 1 to 11, wherein the firstand the second binding members are antibodies or receptors.

Clause 13. The method of any one of clauses 1 to 12, wherein the firstbinding member is a receptor and the second binding member is anantibody or wherein the first binding member is an antibody and thesecond binding member is a receptor.

Clause 14. The method of any one of clauses 1 to 12, wherein the firstbinding member is a first antibody and the second binding member is asecond antibody.

Clause 15. The method of any one of clauses 1 to 14, wherein the tag isnegatively charged and the translocating comprises applying a positivepotential across the layer thereby translocating the tag through thelayer.

Clause 16. The method of any one of clauses 1 to 14, wherein the tag ispositively charged and the translocating comprises applying a negativepotential across the layer thereby translocating the tag through thelayer.

Clause 17. The method of any one of the clauses 1 to 16, wherein atleast the steps (a)-(d) are carried out in a microfluidics device,droplet based microfluidic device; digital microfluidics device (DMF), asurface acoustic wave based microfluidic device (SAW), a fullyintegrated DMF and nanopore device, or a fully integrated SAW andnanopore device.

Clause 18. The method of clauses 17, wherein a DMF element and ananopore element are operatively coupled in the fully integrated DMF andnanopore device, or a SAW element and a nanopore element are operativelycoupled in the fully integrated SAW and nanopore device.

Clause 19. The method of clauses 17, wherein the DMF device or the SAWdevice is fabricated by roll to roll based printed electronics method.

Clause 20. The method of clauses 18, where the DMF element or the SAWelement is fabricated by roll to roll based printed electronic methods.

Clause 21. The method of clauses 17, wherein the fully integrated DMFand nanopore device or the fully integrated SAW and nanopore devicecomprise a microfluidic conduit.

Clause 22. The method of clauses 21, wherein the microfluidic conduitcouples the DMF element to the nanopore element, and the microfluidicconduit comprises a fluidic flow that is induced by passive forces oractive forces.

Clause 23. The method of any one of clauses 1 to 22, wherein thenanopore is a solid state nanopore or a biological nanopore.

Clause 24. The method of any one of the clauses 1 to 23, whereinmeasuring the number of tags translocating through the layer comprisesobserving a change in current induced by an interaction of the tags withthe nanopores.

Clause 25. The method of clauses 24, wherein the analyte is present inthe sample when the current change has a magnitude above a thresholdlevel.

Clause 26. The method of clauses 23, wherein the method furthercomprises transporting a droplet containing the tag obtained in step (d)to a nanopore device and placing the droplet across a nanopore layerpresent in the nanopore device such that the droplet is split by thenanopore layer and is connected by nanopore(s) present in the nanoporelayer, wherein the tag is present in the droplet on both sides of thenanopore layer.

Clause 27. The method of clauses 26, wherein the method comprisestranslocating the tag present on a first side of the nanopore layeracross the nanopore to a second side of the nanopore layer, therebycollecting the tag in the split droplet on the second side of nanoporelayer.

Clause 28. The method of clauses 26, further comprising translocatingthe tag to the first side of the nanopore layer and determining thenumber of tags present in the droplet.

Clause 29. The method of any one of clauses 1 to 28, wherein the tagcomprises a cleavable linker.

Clause 30. A method for measuring or detecting an analyte of interestpresent in a biological sample, the method comprising (a) contacting thesample with a solid support, a first specific binding member, and asecond specific binding member, wherein the solid support comprises animmobilization agent, the first specific binding member comprises aligand for the immobilization agent and the first specific bindingmember specifically binds the analyte of interest, the second specificbinding member comprises a cleavable tag, and the second specificbinding member specifically binds the analyte of interest, wherein asolid support/first specific binding member/analyte of interest/secondspecific binding member complex is formed; (b) removing second specificbinding member not bound to the solid support/first specific bindingmember/analyte/second specific binding member complex; (c) cleaving thetag attached to the labeled analyte bound to the second specific bindingmember in the solid support/first specific binding member/analyte ofinterest/second specific binding member complex; (d) translocating thetag through one or more nanopores in a layer; and (e) assessing the tagstranslocating through the layer, wherein measuring the number of tagstranslocating through the layer measures the amount of analyte presentin the sample, or wherein detecting tags translocating through the layerdetects that the analyte is present in the sample.

Clause 31. A method for measuring or detecting an analyte present in abiological sample, the method comprising: (a) contacting the sample witha first binding member, wherein the first binding member is immobilizedon a solid support and wherein the first binding member specificallybinds to the analyte; (b) contacting the analyte with a second bindingmember, wherein the second binding member specifically binds to theanalyte and wherein the second binding member comprises an aptamer; (c)removing aptamer not bound to the analyte bound to the solid substrate;(d) dissociating the aptamer bound to the analyte (e) translocating thedissociated aptamer through one or more nanopores in a layer; and (f)assessing the aptamer translocating through the layer, wherein measuringthe number of aptamers translocating through the layer measures theamount of analyte present in the sample, or wherein detecting aptamerstranslocating through the layer detects that the analyte is present in athe sample.

Clause 32. The method of clauses 31, wherein each aptamer translocatingthrough the layer is a translocation event and measuring the number oftranslocation events measures the amount of analyte present in thesample, wherein the amount of analyte present in the sample isdetermined by: i) counting the number of translocation events during aset period of time and correlating the number of translocation events toa control; ii) measuring the amount of time for a set number oftranslocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference standardcomprising a calibration curve, standard addition, or digital polymerasechain reaction.

Clause 33. The method of clauses 32, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

Clause 34. The method of any one of clauses 31 to 33, wherein the methodinvolves single molecule counting.

Clause 35. The method of any one of clauses 31 to 34, wherein theaptamer is a DNA aptamer.

Clause 36. The method of any one of clauses 31 to 34, wherein theaptamer is a RNA aptamer.

Clause 37. The method of any one of clauses 31 to 36, wherein the firstbinding member is an antibody.

Clause 38. The method of any one of clauses 31 to 36, wherein theanalyte is a ligand and the first binding member is a receptor.

Clause 39. The method of any one of the clauses 31 to 38, wherein atleast the steps (a)-(d) are carried out in a microfluidics device,droplet based microfluidic device, digital microfluidics device (DMF), asurface acoustic wave based microfluidic device (SAW), a fullyintegrated DMF and nanopore device, or a fully integrated SAW andnanopore device.

Clause 40. The method of clauses 39, wherein a DMF element and ananopore element are operatively coupled in the fully integrated DMF andnanopore device, or a SAW element and a nanopore element are operativelycoupled in the fully integrated SAW and nanopore device.

Clause 41. The method of clauses 39, wherein the DMF device or the SAWdevice is fabricated by roll to roll based printed electronics method.

Clause 42. The method of clauses 40, where the DMF element or the SAWelement is fabricated by roll to roll based printed electronic methods.

Clause 43. The method of clauses 39, wherein the fully integrated DMFand nanopore device or the fully integrated SAW and nanopore devicecomprise a microfluidic conduit.

Clause 44. The method of clauses 43, wherein the microfluidic conduitcouples the DMF element to the nanopore element, and the microfluidicconduit comprises a fluidic flow that is induced by passive forces oractive forces.

Clause 45. The method of any one of clauses 31 to 44, wherein thenanopore is a solid state nanopore or a biological nanopore.

Clause 46. An integrated digital microfluidics nanopore devicecomprising: a first substrate, comprising an array of electrodes; asecond substrate spaced apart from the first substrate; and a nanoporelayer disposed between the first and second substrates, wherein thearray of electrodes are configured to position the droplet across thenanopore layer such that the droplet is split by the nanopore layer intoa first portion and a second portion, wherein at least two electrodes ofthe array of electrodes are positioned across the nanopore layer, wherethe two electrodes form an anode and a cathode and operate to drivecurrent through a nanopore in the nanopore layer when a liquid dropletis positioned across the nanopore layer.

Clause 47. The device of clauses 46, wherein the nanopore layer isattached to the first and second substrates.

Clause 48. The device of clauses 46, wherein the nanopore layer isattached to the first or the second substrate.

Clause 49. The device of any one of clauses 46 to 48, wherein theelectrodes are transparent.

Clause 50. The device of any one of clauses 46 to 49, wherein theelectrodes are disposed in a grid pattern.

Clause 51. The device of any one of clauses 46 to 50, wherein the atleast two electrodes of the array of electrodes positioned across thenanopore layer flank the nanopore layer and are not positioned acrossthe nanopore layer.

Clause 52. The device of any one of clauses 46 to 51, wherein theelectrodes are interdigitated.

Clause 53. The device of any one of clauses 46 to 51, wherein the arrayof electrodes are configured for activation by a power source, whereinthe power source activates the electrodes in a sequential manner.

Clause 54. The device of clauses 53, wherein sequential manner comprisesturned one or more electrodes on or off.

Clause 55. The device of any one of clauses 52 to 54, wherein theactivation of the array of electrodes by a power source is controlled bya set of instructions executed by a processor that controls the powersource.

Clause 56. An integrated digital microfluidics nanopore devicecomprising: a first substrate, comprising an array of electrodes; asecond substrate spaced apart from the first substrate; and a nanoporelayer disposed between the first and second substrates, wherein thearray of electrodes are configured to position a droplet across thenanopore layer such that the nanopore layer splits the droplet into afirst portion and a second portion, wherein at least one electrode ofthe array of electrodes is in contact with the first portion of adroplet positioned across the nanopore layer and the electrode in thesecond substrate is positioned to contact the second portion of thedroplet positioned across the nanopore layer, where the two electrodesform an anode and a cathode and operate to drive current through ananopore in the nanopore layer when a liquid droplet is positionedacross the nanopore layer.

Clause 57. The device of clauses 56, wherein the nanopore layer isattached to the first substrate.

Clause 58. The device of any one of clauses 56 or 57, wherein thenanopore layer is attached to the second substrate.

Clause 59. The device of any one of clauses 56 to 58, wherein the firstand/or second substrates are transparent.

Clause 60. The device of any one of clauses 56 to 59, wherein the arrayof electrodes are transparent.

Clause 61. A kit comprising the device of any one of clauses 46 to 60,or for use in the method of any one of clauses 1 to 45.

Clause 62. The kit of clauses 61, further comprising additionalreagents, wherein at least one reagent comprises a tag than can bedetected by translocation through the nanopore layer of the device.

Clause 63. A method of using the device of any one of clauses 46 to 60,or the method of any one of clauses 1 to 45, for measuring or detectingan analyte present in a biological sample or for diagnosing a patient orscreening a blood supply.

Clause 64. The method of any one of clauses 1 to 45, wherein at leastthe steps (a)-(d) are carried out using the device of any one of clauses46 to 60.

Clause 65. Use of the device of any one of clauses 46 to 60, or the useof the method of any one of clauses 1 to 45, in a method of diagnosing apatient or screening a blood supply or for measuring or detecting ananalyte present in a biological sample.

Clause 66. A method for measuring or detecting an analyte present in abiological sample, the method comprising: (a) contacting the sample witha binding member, wherein the binding member is immobilized on a solidsupport and wherein the binding member specifically binds to theanalyte; (b) contacting the sample with a labeled analyte, wherein thelabeled analyte is labeled with a cleavable tag; (c) removing labeledanalyte not bound to the binding member; (d) cleaving the tag attachedto the labeled analyte bound to the binding member; (e) translocatingthe tag through one or more nanopores in a layer; and (f) assessing thetags translocating through the layer, wherein measuring the number oftags translocating through the layer measures the amount of analytepresent in the sample, or wherein detecting tags translocating throughthe layer detects that the analyte is present in the sample.

Clause 67. A method for measuring or detecting an analyte present in abiological sample, the method comprising: (a) contacting the sample witha binding member, wherein binding member is immobilized on a solidsupport and wherein binding member specifically binds to the analyte;(b) contacting the sample with a labeled analyte, wherein the labeledanalyte comprises an aptamer; (c) removing labeled analyte not bound tothe binding member; (d) dissociating the aptamer bound to the labeledanalyte and translocating the dissociated aptamer through one or morenanopores in a layer; and (e) assessing the aptamer translocatingthrough the layer, wherein measuring the number of aptamerstranslocating through the layer measures the amount of analyte presentin the sample, or wherein detecting aptamers translocating through thelayer detects that the analyte is present in the sample.

Clause 68. A method for measuring or detecting an analyte present in abiological sample, the method comprising: (a) contacting the sample witha binding member, wherein the binding member specifically binds to theanalyte, and the binding member is labeled with a cleavable tag; (b)contacting the sample with a immobilized analyte, wherein theimmobilized analyte is immobilized on a solid support; (c) removingbinding member not bound to the immobilized analyte; (d) cleaving thetag attached to the binding member bound to the immobilized analyte; (e)translocating the tag through one or more nanopores in a layer; and (f)assessing the tag translocating through the layer, wherein measuring thenumber of tags translocating through the layer measures the amount ofanalyte present in the sample, or wherein detecting tags translocatingthrough the layer detects that the analyte is present in the sample.

Clause 69. A method for measuring or detecting an analyte present in abiological sample, the method comprising: (a) contacting the sample witha binding member, wherein the binding member specifically binds to theanalyte, and the binding member comprises an aptamer; (b) contacting thesample with a immobilized analyte, wherein the immobilized analyte isimmobilized on a solid support; (c) removing binding member not bound tothe immobilized analyte; (d) dissociating the aptamer bound to thebinding member bound to the immobilized analyte and translocating thedissociated aptamer through one or more nanopores in a layer; and (e)assessing the aptamer translocating through the layer, wherein measuringthe number of aptamers translocating through the layer measures theamount of analyte present in the sample, or wherein detecting aptamerstranslocating through the layer detects that the analyte is present inthe sample.

Clause 70. The method of clauses 66 or 68, wherein each tagtranslocating through the layer is a translocation event and measuringthe number of translocation events measures the amount of analytepresent in the sample, wherein the amount of analyte present in thesample is determined by: i) counting the number of translocation eventsduring a set period of time and correlating the number of translocationevents to a control; ii) measuring the amount of time for a set numberof translocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference standardcomprising a calibration curve, standard addition, or digital polymerasechain reaction.

Clause 71. The method of clauses 67 or 69, wherein each aptamertranslocating through the layer is a translocation event and measuringthe number of translocation events measures the amount of analytepresent in the sample, wherein the amount of analyte present in thesample is determined by: i) counting the number of translocation eventsduring a set period of time and correlating the number of translocationevents to a control; ii) measuring the amount of time for a set numberof translocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference standardcomprising a calibration curve, standard addition, or digital polymerasechain reaction.

Clause 72. The method of clauses 70 or 71, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

Clause 73. The method of any one of clauses 66 to 72, wherein the methodinvolves single molecule counting.

Clause 74. The method of any one of clauses 66, 68, 70, 72, or 73,wherein at least the steps (a)-(d) are carried out using the device ofany one of clauses 46 to 60.

Clause 75. The method of any one of clauses 67, 69, 71, 72, or 73,wherein at least the steps (a)-(d) are carried out using the device ofany one of clauses 46 to 60.

Clause 76. The method of any one of clauses 66, 68, 70, 72, 73, or 74,wherein the tag is selected from the group consisting of an anionicpolymer, a cationic polymer, a dendrimer, and a nanoparticle.

Clause 77. The method of any one of clauses 66, 68, 70, 72, 73, or 74,wherein the tag is substantially spherical or hemi-spherical.

Clause 78. The method of any one of clauses 66, 68, 70, 72, 73, or 74,wherein the tag is substantially spherical and comprises a nanoparticle.

Clause 79. The method of any one of clauses 66, 68, 70, 72, 73, or 74,wherein the tag is substantially spherical or hemi-spherical andcomprises a dendrimer.

Clause 80. The method of clauses 79, wherein the dendrimer is positivelyor negatively charged.

Clause 81. The method of clauses 79, wherein the nanoparticle comprisesa positively charged nanoparticle.

Clause 82. The method of clauses 76 or 78, wherein the nanoparticlecomprises a negatively charged nanoparticle.

Clause 83. The method of any one of clauses 66, 68, 70, 72, 73, 74, or76 to 82, wherein the binding member is an antibody or receptor.

Clause 84. The method of any one of clauses 66, 68, 70, 72, 73, 74, or76 to 83, wherein the tag is negatively charged and the translocatingcomprises applying a positive potential across the layer therebytranslocating the tag across the layer.

Clause 85. The method of any one of clauses 66, 68, 70, 72, 73, 74, or76 to 84, wherein the tag is positively charged and the translocatingcomprises applying a negative potential across the layer therebytranslocating the tag across the layer.

Clause 86. The method of any one of the clauses 66, 68, 70, 72, 73, 74,or 76 to 85, wherein measuring the number of tags translocating throughthe layer comprises observing a current blockade effect of the tags onthe nanopores.

Clause 87. The method of clauses 86, wherein the analyte is present inthe sample when the current blockade effect is above a threshold level.

Clause 88. The method of any one of clauses 67, 69, 71, 72, 73, or 75,wherein the aptamer is a DNA aptamer.

Clause 89. The method of any one of clauses 67, 69, 71, 72, 73, or 75,wherein the aptamer is a RNA aptamer.

Clause 90. The method of any one of clauses 67, 69, 71, 72, 73, 75, 88,or 89, wherein the binding member is an antibody.

Clause 91. The method of any one of clauses 67, 69, 71, 72, 73, 75, 88,or 89, wherein the analyte is a ligand and the binding member is areceptor.

Clause 92. The method of any one of clauses 67, 69, 71, 72, 73, 75, or88 to 91, wherein the method further comprises transporting a dropletcontaining the tag to a nanopore device and placing the droplet across ananopore layer present in the nanopore device such that the droplet issplit by the nanopore layer and is connected by nanopore(s) present inthe nanopore layer, wherein the tag is present in the droplet on bothsides of the nanopore layer.

Clause 93. The method of clauses 92, wherein the method comprisestranslocating the tag present on a first side of the nanopore layeracross the nanopore to a second side of the nanopore layer, therebycollecting the tag in the split droplet on the second side of nanoporelayer

Clause 94. The method of clauses 92, further comprising translocatingthe tag to the first side of the nanopore layer and determining thenumber of tags present in the droplet.

Clause 95. The method of any one of clauses 67, 69, 71, 72, 73, 75, or88 to 94, wherein the method further comprises transporting a dropletcontaining the aptamer to a nanopore device and placing the dropletacross a nanopore layer present in the nanopore device such that thedroplet is split by the nanopore layer and is connected by nanopore(s)present in the nanopore layer, wherein the aptamer is present in thedroplet on both sides of the nanopore layer.

Clause 96. The method of clauses 95, wherein the method comprisestranslocating the aptamer present on a first side of the nanopore layeracross the nanopore to a second side of the nanopore layer, therebycollecting the aptamer in the split droplet on the second side ofnanopore layer

Clause 97. The method of clauses 95, further comprising translocatingthe aptamer to the first side of the nanopore layer and determining thenumber of aptamers present in the droplet.

Clause 98. The method of any one of clauses 66 to 97, wherein thenanopore is a solid state nanopore or a biological nanopore.

Clause 99. The method of any one of clauses 1 to 45 or 63 to 98, whereinthe second binding member further comprises a spacer.

Clause 100. The method of clauses 99, wherein the spacer comprises anitrobenzyl group, dithioethylamino, 6 carbon spacer, 12 carbon spacer,or3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonate.

Clause 101. The method of clauses 100, wherein the spacer comprises anitrobenzyl group, and the tag is a DNA molecule.

Clause 102. The method of clauses 100, wherein the spacer isdithioethylamino and the tag is a carboxylated nanoparticle.

Clause 103. The method of clauses 100, wherein the spacer is3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonateand the tag is an oligonucleotide.

Clause 104. The method of clauses 103, wherein the spacer comprises a 6carbon spacer or a 12 carbon spacer and the tag is biotin.

Clause 105. The method of clauses 104, wherein the second binding membercomprises a nucleic acid comprising a nucleotide sequence set forth inany one of SEQ ID NOs: 1-11.

Clause 106. An integrated digital microfluidics nanopore devicecomprising a microfluidics module and a nanopore module; themicrofluidics module comprising an array of electrodes, wherein thearray of electrodes transports at least one droplet of fluid to a firsttransfer position in the array of electrodes, wherein the first transferposition is at an interface between the microfluidics module and thenanopore module; the nanopore module comprising: a first capillarychannel; and a second capillary channel; wherein at least the firstcapillary channel extends to the interface and is adjacent to the firsttransfer position, and is positioned to receive a fluid dropletpositioned at the first transfer position; wherein the first capillarychannel intersects with the second capillary channel, wherein a nanoporelayer is positioned in between the first and second capillary channelsat the location where the first and the second capillary channelsintersect.

Clause 107. The device of clauses 106, wherein the array of electrodestransports at least one droplet of fluid to a second transfer positionin the array of electrodes, wherein the second transfer position is atan interface between the microfluidics module and the nanopore modulewherein the second capillary channel extends to the interface and isadjacent to the second transfer position, and is positioned to receive afluid droplet positioned at the second transfer position.

Clause 108. The device of clauses 106, wherein the second capillarychannel extends between a vent or a reservoir on one or both ends of thesecond capillary channel.

Clause 109. The device of clauses 108, wherein the second capillarychannel is connected to a first reservoir at one end and a secondreservoir at the other end.

Clause 110. The device of clauses 109, wherein the first reservoirand/or the second reservoir comprises a fluid to be positioned withinthe second capillary channel at the intersection which fluid facilitatesoperation of the nanopore layer to drive current through a nanopore ofthe nanopore layer.

Clause 111. The device of clauses 109, wherein the first capillarychannel and/or the second capillary channel varies in cross sectionalwidth across a length of the capillary channel such that the widthdecreases at the intersection compared to the width on either sides ofthe intersection.

Clause 112. The device of clauses 106, wherein the first capillarychannel comprises a first pair of electrodes and the second capillarychannel comprises a second pair of electrodes, wherein the first pair ofelectrodes is positioned in the first capillary channel and flank thenanopore in the nanopore layer and wherein second pair of electrodes ispositioned in the second capillary channel and flank the nanopore in thenanopore layer.

Clause 113. The device of any one of clauses 107 to 112, wherein thedroplets are droplets comprising a molecule to be counted bytransporting through the nanopore in the nanopore layer.

Clause 114. The device of clauses 107, wherein the fluid droplets havedifferent compositions and are a first droplet and a second droplet, thefirst droplet comprising a molecule to be counted by transporting acrossthe nanopore layer through the nanopore and the second dropletcomprising a conductive fluid lacking the molecule, where the conductivefluid facilitates transport of the molecule across the nanopore layervia the nanopore.

Clause 115. The device of any one of clauses 106 to 114, wherein thefirst capillary channel comprises a first electrode positioned proximalto the nanopore layer and the second capillary channel comprising asecond electrode positioned proximal to the nanopore layer, wherein eachof the first and second electrodes are exposed in the capillary channelssuch that they are in contact with a fluid present in the capillarychannels and wherein the first and second electrodes operate to drivecurrent through a nanopore in the nanopore layer when a liquid ispositioned across the nanopore layer in the first and second capillarychannels.

Clause 116. The device of any one of clauses 106 to 115, wherein thefirst transfer position and the first capillary channel are onsubstantially the same plane, and wherein the fluid droplet is alignedwith an opening of the first capillary channel.

Clause 117. The device of any one of clauses 106 to 115, wherein thefirst transfer position is at a plane higher than the first capillarychannel and wherein the device is configured with a vertical port fortransferring the fluid droplet down to an opening of the first capillarychannel.

Clause 118. The device of clauses 117, wherein the first surface of thefirst substrate comprises a first area on which the array of electrodesis disposed and a second area in which the first microchannel is formed,wherein the array of electrodes is on a plane higher than the plane atwhich the first microchannel is formed.

Clause 119. The device of clauses 117, wherein the second substratecomprises a notch at a side edge located at the interface, wherein thenotch is aligned over the first capillary channel and provides avertical port for transport of a droplet located at the transferelectrode to the opening of the first capillary channel.

Clause 120. The device of any one of clauses 106 to 119, furthercomprising a single electrode spaced apart from the array of electrodes,wherein the single electrode extends over at least a portion of thearray of electrodes at the first transfer position and is in bi-planarconfiguration with the at least a portion of the array of electrodes atthe first transfer position.

Clause 121. The device of any one of clauses 107 to 119, furthercomprising a single electrode spaced apart from the array of electrodes.

Clause 122. The device of any one of clauses 106 to 119, furthercomprising a single electrode spaced apart from the array of electrodes,wherein the single electrode does not extend over the first transferposition and is not in bi-planar configuration with the array ofelectrodes, wherein the fluid droplet is moved to the first transferposition by using coplanar electrodes.

Clause 123. The device of any one of clauses 106 to 119, furthercomprising a single electrode spaced apart from the array of electrodeswherein the single electrode does not extend over the first transferposition and is not in bi-planar configuration with the array ofelectrodes, wherein the fluid droplets are moved to the transferposition by using coplanar electrodes.

Clause 124. A method of using the device of any one of clauses 106 to123, or the method of any one of clauses 66 to 105, for measuring ordetecting an analyte present in a biological sample or for diagnosing apatient or screening a blood supply.

Clause 125. The method of any one of clauses 1 to 45 or 66 to 105,wherein at least the steps (a)-(d) are carried out using the device ofany one of clauses 106 to 123.

Clause 126. Use of the device of any one of clauses 106 to 123, or theuse of the method of any one of clauses 1 to 45, in a method ofdiagnosing a patient or screening a blood supply or for measuring ordetecting an analyte present in a biological sample.

Clause 127. A method for measuring an analyte present in a biologicalsample, the method comprising: (a) contacting the sample with a firstbinding member, wherein the first binding member is immobilized on asolid support and wherein the first binding member specifically binds tothe analyte; (b) contacting the analyte with a second binding member,wherein the second binding member specifically binds to the analyte andwherein the second binding member comprises a cleavable tag attachedthereto; (c) removing second binding member not bound to the analytebound to the first binding member; (d) cleaving the tag attached to thesecond binding member bound to the analyte bound to the first bindingmember; (e) translocating the tag through one or more nanopores in alayer; and (f) assessing the tag translocating through the layer,wherein each tag translocating through the layer is a translocationevent, wherein measuring the number of translocation events measures theamount of analyte present in the sample, wherein the amount of analytepresent in the sample is determined by: i) counting the number oftranslocation events during a set period of time and correlating thenumber of translocation events to a control; ii) measuring the amount oftime for a set number of translocation events to occur and correlatingto a control; or iii) measuring the average time between translocationevents to occur and correlating to a control, wherein the control is areference standard comprising a calibration curve, standard addition, ordigital polymerase chain reaction, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

Clause 128. A method for measuring an analyte present in a biologicalsample, the method comprising: (a) contacting the sample with a firstbinding member, wherein the first binding member is immobilized on asolid support and wherein the first binding member specifically binds tothe analyte; (b) contacting the analyte with a second binding member,wherein the second binding member specifically binds to the analyte andwherein the second binding member comprises an aptamer; (c) removingaptamer not bound to the analyte bound to the solid substrate; (d)dissociating the aptamer bound to the analyte and (e) translocating thedissociated aptamer through one or more nanopores in a layer; and (f)assessing the aptamer translocating through the layer, wherein eachaptamer translocating through the layer is a translocation event,wherein measuring the number of translocation events measures the amountof analyte present in the sample, wherein the amount of analyte presentin the sample is determined by: i) counting the number of translocationevents during a set period of time and correlating the number oftranslocation events to a control; ii) measuring the amount of time fora set number of translocation events to occur and correlating to acontrol; or iii) measuring the average time between translocation eventsto occur and correlating to a control, wherein the control is areference standard comprising a calibration curve, standard addition, ordigital polymerase chain reaction, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

Clause 129. A method for measuring an analyte present in a biologicalsample, the method comprising: (a) contacting the sample with a bindingmember, wherein the binding member is immobilized on a solid support andwherein the binding member specifically binds to the analyte; (b)contacting the sample with a labeled analyte, wherein the labeledanalyte is labeled with a cleavable tag; (c) removing labeled analytenot bound to the binding member; (d) cleaving the tag attached to thelabeled analyte bound to the binding member; (e) translocating the tagthrough one or more nanopores in a layer; and (f) assessing the tagstranslocating through the layer, wherein each tag translocating throughthe layer is a translocation event, wherein measuring the number oftranslocation events measures the amount of analyte present in thesample, wherein the amount of analyte present in the sample isdetermined by: i) counting the number of translocation events during aset period of time and correlating the number of translocation events toa control; ii) measuring the amount of time for a set number oftranslocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference standardcomprising a calibration curve, standard addition, or digital polymerasechain reaction, wherein the standard curve in subsection i) isdetermined by measuring the number of translocation events for controlconcentrations of analyte during a set period of time; wherein thestandard curve in subsection ii) is determined by measuring the time ittakes for a set number of translocation events to occur for controlconcentrations of analyte; and wherein the standard curve in subsectioniii) is determined by measuring the average time between translocationevents to occur for control concentrations of analyte.

Clause 130. A method for measuring an analyte present in a biologicalsample, the method comprising: (a) contacting the sample with a bindingmember, wherein binding member is immobilized on a solid support andwherein binding member specifically binds to the analyte; (b) contactingthe sample with a labeled analyte, wherein the labeled analyte comprisesan aptamer; (c) removing labeled analyte not bound to the bindingmember; (d) dissociating the aptamer bound to the labeled analyte andtranslocating the dissociated aptamer through one or more nanopores in alayer; and (e) assessing the aptamer translocating through the layer,wherein each aptamer translocating through the layer is a translocationevent, wherein measuring the number of translocation events measures theamount of analyte present in the sample, wherein the amount of analytepresent in the sample is determined by: i) counting the number oftranslocation events during a set period of time and correlating thenumber of translocation events to a control; ii) measuring the amount oftime for a set number of translocation events to occur and correlatingto a control; or iii) measuring the average time between translocationevents to occur and correlating to a control, wherein the control is areference standard comprising a calibration curve, standard addition, ordigital polymerase chain reaction, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

Clause 131. A method for measuring an analyte present in a biologicalsample, the method comprising: (a) contacting the sample with a bindingmember, wherein the binding member specifically binds to the analyte,and the binding member is labeled with a cleavable tag; (b) contactingthe sample with a immobilized analyte, wherein the immobilized analyteis immobilized on a solid support; (c) removing binding member not boundto the immobilized analyte; (d) cleaving the tag attached to the bindingmember bound to the immobilized analyte; (e) translocating the tagthrough one or more nanopores in a layer; and (f) assessing the tagtranslocating through the layer, wherein each tag translocating throughthe layer is a translocation event, wherein measuring the number oftranslocation events measures the amount of analyte present in thesample, wherein the amount of analyte present in the sample isdetermined by: i) counting the number of translocation events during aset period of time and correlating the number of translocation events toa control; ii) measuring the amount of time for a set number oftranslocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference standardcomprising a calibration curve, standard addition, or digital polymerasechain reaction, wherein the standard curve in subsection i) isdetermined by measuring the number of translocation events for controlconcentrations of analyte during a set period of time; wherein thestandard curve in subsection ii) is determined by measuring the time ittakes for a set number of translocation events to occur for controlconcentrations of analyte; and wherein the standard curve in subsectioniii) is determined by measuring the average time between translocationevents to occur for control concentrations of analyte.

Clause 132. A method for measuring an analyte present in a biologicalsample, the method comprising: (a) contacting the sample with a bindingmember, wherein the binding member specifically binds to the analyte,and the binding member comprises an aptamer; (b) contacting the samplewith a immobilized analyte, wherein the immobilized analyte isimmobilized on a solid support; (c) removing binding member not bound tothe immobilized analyte; (d) dissociating the aptamer bound to thebinding member bound to the immobilized analyte and translocating thedissociated aptamer through one or more nanopores in a layer; and (e)assessing the aptamer translocating through the layer, wherein eachaptamer translocating through the layer is a translocation event,wherein measuring the number of translocation events measures the amountof analyte present in the sample, wherein the amount of analyte presentin the sample is determined by: i) counting the number of translocationevents during a set period of time and correlating the number oftranslocation events to a control; ii) measuring the amount of time fora set number of translocation events to occur and correlating to acontrol; or iii) measuring the average time between translocation eventsto occur and correlating to a control, wherein the control is areference standard comprising a calibration curve, standard addition, ordigital polymerase chain reaction, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

Clause 133. A method for measuring or detecting an analyte present in abiological sample, the method comprising: (a) contacting the sample witha binding member, wherein the binding member is immobilized on a solidsupport, the binding member comprises a cleavable tag attached thereto,and the binding member specifically binds to the analyte; (b) removingbinding member not bound to the analyte; (c) cleaving the tag attachedto the binding member bound to the analyte; (d) translocating the tagthrough one or more nanopores in a layer; and (e) assessing the tagtranslocating through the layer, wherein each tag translocating throughthe layer is a translocation event, wherein measuring the number oftranslocation events measures the amount of analyte present in thesample, wherein the amount of analyte present in the sample isdetermined by: i) counting the number of translocation events during aset period of time and correlating the number of translocation events toa control; ii) measuring the amount of time for a set number oftranslocation events to occur and correlating to a control; or iii)measuring the average time between translocation events to occur andcorrelating to a control, wherein the control is a reference comprisinga calibration curve, standard addition, or digital polymerase chainreaction.

Clause 134. The method of clauses 133, wherein the standard curve insubsection i) is determined by measuring the number of translocationevents for control concentrations of analyte during a set period oftime; wherein the standard curve in subsection ii) is determined bymeasuring the time it takes for a set number of translocation events tooccur for control concentrations of analyte; and wherein the standardcurve in subsection iii) is determined by measuring the average timebetween translocation events to occur for control concentrations ofanalyte.

Clause 135. An integrated digital microfluidics nanopore-enabled devicecomprising: a microfluidics module and a nanopore-enabled module; themicrofluidics module, comprising an array of electrodes spaced apartfrom a single electrode sized to overlap with at least a portion of thearray of electrodes, where the array of electrodes and the singleelectrode transport at least one droplet of fluid to a transferelectrode in the array of electrodes, wherein the transfer electrode ispositioned at an interface between the microfluidics module and thenanopore-enabled module; the nanopore-enabled module comprising: a firstmicrochannel positioned on a first surface of a first substrate; asecond microchannel positioned on a first surface of a second substrate;wherein the first surface of the first substrate is in contact with thefirst surface of the second substrate thereby enclosing the firstmicrochannel and the second microchannel to provide a first capillarychannel and a second capillary channel, respectively, wherein at leastthe first capillary channel extends to the interface between themicrofluidics module and the nanopore-enabled module and is adjacent tothe transfer electrode, and is positioned to receive a fluid dropletpositioned on the transfer electrode; wherein the first capillarychannel intersects with the second capillary channel, wherein a layer ispositioned in between the first and second substrates at the locationwhere the first and the second capillary channels intersect, wherein thelayer is devoid of a nanopore and separates an ionic liquid present inthe first and second capillary channels, wherein the first and secondcapillary channels are in electrical connection with electrodes fordriving a voltage from the first to the second capillary channel or viceversa for creating a nanopore in the layer at the intersection of thefirst and second capillary channels.

Clause 136. The device of clauses 135, wherein the ionic liquid is anaqueous solution.

Clause 137. The device of clauses 136, wherein the aqueous solution issalt solution.

Clause 138. The device of any one of clauses 135 to 137, wherein theionic liquid comprises an analyte of interest, wherein the device isconfigured to detect the presence or absence of the analyte in the ionicliquid.

Clause 139. A method for generating a nanopore in an integrated digitalmicrofluidics nanopore-enabled device, the method comprising: providingan integrated digital microfluidics nanopore-enabled device of any oneof clauses 135 to 138; applying a voltage in the first and secondcapillary channels to drive current through the layer; measuringconductance across the layer; terminating application of voltage upondetection of a conductance indicative of generation of a nanopore in thelayer.

Clause 140. An integrated digital microfluidics nanopore devicecomprising: a first substrate comprising an array of electrodes; asecond substrate spaced apart from the first substrate; an opening inthe first or second substrate in fluid communication with a nanoporelayer comprising a nanopore; and a pair of electrodes configured toapply an electric field through the nanopore, wherein the array ofelectrodes are configured to transport at least one droplet of fluid tothe opening.

Clause 141. The device of clauses 140, wherein the opening is acapillary channel.

Clause 142. The device of clauses 141, wherein the capillary channel hasan opening on a first side of the first or second substrate that iswider than an opening on a second side of the first or second substrate.

Clause 143. The device of clauses 142, wherein the pair of detectionelectrodes comprises a first detection electrode that is the singleelectrode.

Clause 144. The device of clauses 142 or 143, wherein the pair ofdetection electrodes comprises a second detection electrode disposed onthe second side.

Clause 145. A pair of integrated digital microfluidics nanopore devicescomprising:

a first integrated digital microfluidics nanopore device according toclauses 142, wherein the single electrode is a first single electrode,and the capillary channel is a first capillary channel; and a secondintegrated digital microfluidics nanopore device comprising: a thirdsubstrate, comprising a fifth side and a sixth side opposite the fifthside, wherein the fifth side comprises an array of electrodes; a fourthsubstrate spaced apart from the third substrate, wherein the fourthsubstrate comprises a seventh side facing the fifth side of the thirdsubstrate and a eight side opposite the seventh side, wherein theseventh side comprises a second single electrode and wherein thenanopore layer is disposed on the eight side, wherein the fourthsubstrate comprises a second capillary channel extending from theseventh side to the eight side of the fourth substrate, wherein thenanopore layer is positioned over an opening of the capillary channel,wherein the nanopore layer is interposed between the second substrateand the fourth substrate such that the nanopore provides anelectroosmotic conduit between the first capillary channel and thesecond capillary channel, wherein the pair of detection electrodescomprises a second detection electrode that is the second singleelectrode.

Clause 146. An integrated digital microfluidics nanopore-enabled devicecomprising: a first substrate, comprising a first side and a second sideopposite the first side, wherein the first side comprises an array ofelectrodes; a second substrate spaced apart from the first substrate,wherein the second substrate comprises a third side facing the firstside of the first substrate and a fourth side opposite the third side; ananopore-enabled layer devoid of a nanopore and disposed on an externalside of the device, wherein the external side is selected from thesecond side or the fourth side, wherein one of the first or secondsubstrates comprising the external side comprises a capillary channelextending from the first side to the second side of the first substrate,or the third side to the fourth side of the second substrate, whereinthe nanopore-enabled layer is positioned over an opening of thecapillary channel; and a pair of electrodes configured to apply anelectric field across the nanopore-enabled layer, wherein the array ofelectrodes are configured to transport at least one droplet of fluid tothe capillary channel.

Clause 147. A method for generating a nanopore in an integrated digitalmicrofluidics nanopore-enabled device, the method comprising: providingan integrated digital microfluidics nanopore-enabled device of clauses143; submerging both sides of the nanopore-enabled layer in an ionicliquid such that the ionic liquid on each side of the layer is inelectrical contact with either one of the pair of detection electrodes;applying a voltage between the pair of detection electrodes to drivecurrent through the layer; measuring conductance across the layer;terminating application of voltage upon detection of a conductanceindicative of generation of a nanopore in the layer.

Clause 148. The method of clauses 147, wherein the ionic liquid is asalt solution.

Clause 149. The method of clauses 147 or 148, wherein the ionic liquidcomprises an analyte of interest, wherein the device is configured todetect the presence or absence of the analyte in the ionic liquid.

Clause 150. The method of any one of clauses 139 or 147 to 149, furthercomprising conditioning the generated nanopore.

Clause 151. The method of clauses 150, wherein the conditioningcomprises: alternately applying a first voltage having a first polarityand a second voltage having a second polarity opposite the firstpolarity across the nanopore membrane, wherein the first and secondvoltages are each applied at least once; and measuring an electroosmoticproperty related to a size of the nanopore.

Clause 152. The method of clauses 150 or 151, further comprisingmeasuring the electroosmotic property related to a size of the nanoporebefore the conditioning.

Clause 153. A composition comprising a binding member, a tag and aspacer.

Clause 154. The composition of clauses 153, wherein the spacer comprisesa nitrobenzyl group, dithioethylamino, 6 carbon spacer, 12 carbonspacer, or3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonate.

Clause 155. The composition of 154, wherein the spacer comprises anitrobenzyl group, and the tag is a DNA molecule.

Clause 156. The composition of 154, wherein the spacer isdithioethylamino and the tag is a carboxylated nanoparticle.

Clause 157. The composition of 154, wherein the spacer is3-(9-((3-carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1-sulfonateand the tag is an oligonucleotide.

Clause 158. The composition of 154, wherein the spacer comprises a 6carbon spacer or 12 carbon spacer and the tag is biotin.

Clause 159. The composition of 158, wherein the second binding membercomprises a nucleic acid comprising a nucleotide sequence set forth inany one of SEQ ID NOs: 1-11.

Clause 160. The composition of any one of clauses 153-159, wherein thetag comprises a cleavable linker.

Clause 161. The composition of clauses 160, wherein the cleavable linkeris selected from the group consisting of a photocleavable linker, achemically cleavable linker, a thermally cleavable linker, athermal-sensitive cleavable linker, and an enzymatic cleavable linker.

Clause 162. The composition of clauses 161, wherein the cleavable linkeris a photocleavable linker, wherein the photocleavable linker comprisinga photocleavable moiety derived from

Clause 163. The composition of clauses 161, wherein the cleavable linkeris a thermally cleavable linker and is cleaved using localizedtemperature elevation.

Clause 164. The composition of clauses 163, wherein the localizedtemperature elevation is generated photothermally or by microwaveirradiation.

Clause 165. The composition of clauses 164, wherein energy from light istransferred to an absorbing target.

Clause 166. The composition of clauses 165, wherein the absorbing targetcomprises a dye, pigment, or water.

Clause 167. The composition of any one of clauses 163 to 166, whereinthe cleavable linker comprises double stranded DNA.

Clause 168. The composition of clauses 161, wherein the cleavable linkeris a chemically cleavable linker and cleavage is mediated by thiol.

Clause 169. The method of any one of clauses 29, 66, 68, 70, 72 to 86,92-94, 98 to 105 127, 129, 131, 133, and 134, wherein the tag comprisesa cleavable linker.

Clause 170. The method of clauses 169, wherein the cleavable linker isselected from the group consisting of a photocleavable linker, achemically cleavable linker, a thermally cleavable linker, athermal-sensitive cleavable linker, and an enzymatic cleavable linker.

Clause 171. The method of clauses 170, wherein the cleavable linker is aphotocleavable linker and the photocleavable linker comprises aphotocleavable moiety derived from

Clause 172. The method of clauses 170, wherein the cleavable linker is athermally cleavable linker and is cleaved using localized temperatureelevation.

Clause 173. The method of clauses 172, wherein the localized temperatureelevation is generated photothermally or by microwave irradiation.

Clause 174. The method of clauses 173, wherein energy from light istransferred to an absorbing target.

Clause 175. The method of clauses 174, wherein the absorbing targetcomprises a dye, pigment, or water.

Clause 176. The method of any one of clauses 172 to 175, wherein thecleavable linker comprises double stranded DNA.

Clause 177. The method of clauses 30 and 170, wherein the cleavablelinker is a chemically cleavable linker and is cleaved by thiol.

Clause 178. The method of any one of clauses 2-30, 31-45, 64, 70 to 105,125, 127 to 134, and 169 to 177, wherein one or more translocationevents corresponds to a binding event of a binding member to an analyte.

Clause 179. The method of clauses 178, wherein one translocation eventcorresponds to a binding event of a binding member to an analyte.

Clause 180. The method of clauses 178, wherein two or more translocationevents corresponds to a binding event of a binding member to an analyte.

Clause 181. The method of clauses 180, wherein two or more tags areincorporated per binding member and two or more translocation eventsrepresents the binding of the binding member to the analyte.

Clause 182. The method of any one of clauses 1 to 45, 63 to 105, 124,125, 127 to 134, and 169 to 181, wherein at least two or more nanoporesare in the layer.

Clause 183. The method of clauses 182, wherein the at least two or morenanopores are presented side by side or in series.

Clause 184. An integrated digital microfluidics nanopore devicecomprising: a first substrate, comprising an array of electrodes; asecond substrate spaced apart from the first substrate; and a nanoporelayer having a first surface and a second surface disposed between thefirst and second substrates, wherein the array of electrodes areconfigured to position a first droplet at the first surface of thenanopore layer, wherein at least two electrodes of the array ofelectrodes are positioned across the nanopore layer, where the twoelectrodes form an anode and a cathode and operate to drive currentthrough a nanopore in the nanopore layer when a liquid droplet is at thefirst surface of the nanopore layer.

Clause 185. The array of electrodes of clauses 173 is further configuredto position a second droplet at a second surface of the nanopore layer.

Clause 186. An integrated digital microfluidics nanopore devicecomprising a microfluidics module and a nanopore module; themicrofluidics module comprising an array of electrodes, where the arrayof electrodes transport at least one droplet of fluid to a transferposition in the array of electrodes, wherein the transfer position is atan interface between the microfluidics module and the nanopore module;the nanopore module comprising: a first capillary channel extending fromthe transfer position to a nanopore layer.

Clause 187. An integrated digital microfluidics nanopore devicecomprising: a first substrate, comprising an array of electrodes; asecond substrate spaced apart from the first substrate; a first nanoporelayer having one or more nanopores therein; a second nanopore layerhaving one or more nanopores therein; and at least two electrodes forcreating an electric field to drive tags through a nanopore in the firstand second nanopore layers.

Clause 188. The method of clauses 30, wherein the immobilization agentcomprises biotin or streptavidin.

Clause 189. The method of clauses 188, wherein the immobilization agentcomprises biotin and ligand comprises streptavidin.

Clause 190. The method of clauses 188, wherein the immobilization agentcomprises streptavidin and ligand comprises biotin.

Clause 191. The method of any one of clauses 30 and 188 to 190, whereinthe solid support, the first binding member, and second binding memberare added sequentially or simultaneously to the sample.

Clause 192. The method of any one of clauses 1 to 45, 63 to 105, 124,125, 127 to 134, 169 to 181, 183, and 188 to 191 wherein the ratio ofthe size of the pore to the tag is at or less than 1.0.

Clause 193. A method for measuring or detecting an analyte of interestpresent in a biological sample, the method comprising (a) contacting thesample with a solid support, a binding member, and a labeled analytethat is labeled with a cleavable tag, wherein the solid supportcomprises an immobilization agent, the binding member comprises a ligandfor the immobilization agent, and the binding member specifically bindsthe analyte of interest so as to form either a solid support/bindingmember/analyte of interest complex or a solid support/bindingmember/labeled analyte complex; (b) removing labeled analyte not boundto the binding member in the solid support/binding member/labeledanalyte complex; (c) cleaving the tag attached to the labeled analytebound to the binding member in the solid support/binding member/labeledanalyte complex; (d) translocating the tag through one or more nanoporesin a layer; and (e) assessing the tags translocating through the layer,wherein measuring the number of tags translocating through the layermeasures the amount of analyte present in the sample, or whereindetecting tags translocating through the layer detects that the analyteis present in the sample.

Clause 194. The method of clause 193, wherein the immobilization agentcomprises biotin or streptavidin.

Clause 195. The method of clause 194, wherein the immobilization agentcomprises biotin and ligand comprises streptavidin.

Clause 196. The method of clause 194, wherein the immobilization agentcomprises streptavidin and ligand comprises biotin.

Clause 197. The method of any one of clauses 193 to 196, wherein thesolid support, the binding member, and the labeled analyte are addedsequentially or simultaneously to the sample.

Clause 198. A method for measuring or detecting an analyte of interestpresent in a biological sample, the method comprising (a) contacting thesample with a solid support, a binding member, and exogenous analyte,wherein the solid support comprises an immobilization agent, theexogenous analyte comprises a ligand for the immobilization agent andbinds the solid support so as to form a solid support/immobilizedanalyte complex, and the binding member comprises a cleavable tag andspecifically binds the analyte of interest so as to form either a solidsupport/analyte of interest/binding member complex or a solidsupport/immobilized analyte/binding member complex; (b) removing bindingmember not bound in either the solid support/immobilized analyte/bindingmember complex or the solid support/analyte of interest/binding membercomplex; (c) cleaving the tag attached to the binding member in thesolid support/immobilized analyte/binding member complex; (d)translocating the tag through one or more nanopores in a layer; and (e)assessing the tags translocating through the layer, wherein measuringthe number of tags translocating through the layer measures the amountof analyte present in the sample, or wherein detecting tagstranslocating through the layer detects that the analyte is present inthe sample.

Clause 199. The method of clause 198, wherein the immobilization agentcomprises biotin or streptavidin.

Clause 200. The method of clause 199, wherein the immobilization agentcomprises biotin and ligand comprises streptavidin.

Clause 201. The method of clause 199, wherein the immobilization agentcomprises streptavidin and ligand comprises biotin.

Clause 202. The method of any one of clauses 198 to 201, wherein thesolid support, the binding member, and exogenous analyte are addedsequentially or simultaneously to the sample.

Clause 203. The method of any one of clauses 198 to 201, wherein priorto cleaving the tag in step (c), the solid support/immobilizedanalyte/binding member complex is isolated.

Clause 204. The method of clause 203 wherein isolation can be done withuse of a magnetic field.

Clause 205. A method for measuring or detecting an analyte of interestpresent in a biological sample, the method comprising (a) contacting thesample with a solid support, a binding member, and a labeled analytethat is labeled with an aptamer, wherein the solid support comprises animmobilization agent, the binding member comprises a ligand for theimmobilization agent, and the binding member specifically binds theanalyte of interest so as to form either a solid support/bindingmember/analyte of interest complex or a solid support/bindingmember/labeled analyte complex; (b) removing labeled analyte not boundto the binding member in the solid support/binding member/labeledanalyte complex; (c) dissociating the aptamer attached to the labeledanalyte bound to the binding member in the solid support/bindingmember/labeled analyte complex; (d) translocating the dissociatedaptamer through one or more nanopores in a layer; and (e) assessing theaptamer translocating through the layer, wherein measuring the number ofaptamers translocating through the layer measures the amount of analytepresent in the sample, or wherein detecting aptamers translocatingthrough the layer detects that the analyte is present in the sample.

Clause 206. The method of clause 205, wherein the immobilization agentcomprises biotin or streptavidin.

Clause 207. The method of clause 206, wherein the immobilization agentcomprises biotin and ligand comprises streptavidin.

Clause 208. The method of clause 206, wherein the immobilization agentcomprises streptavidin and ligand comprises biotin.

Clause 209. The method of any one of clauses 205 to 208, wherein thesolid support, the binding member, and the labeled analyte are addedsequentially or simultaneously to the sample.

Clause 210. A method for measuring or detecting an analyte of interestpresent in a biological sample, the method comprising (a) contacting thesample with a solid support, a binding member, and exogenous analyte,wherein the solid support comprises an immobilization agent, theexogenous analyte comprises a ligand for the immobilization agent andbinds the solid support so as to form a solid support/immobilizedanalyte complex, and the binding member comprises an aptamer andspecifically binds the analyte of interest so as to form either a solidsupport/analyte of interest/binding member complex or a solidsupport/immobilized analyte/binding member complex; (b) removing bindingmember not bound in either the solid support/immobilized analyte/bindingmember complex or the solid support/analyte of interest/binding membercomplex; (c) dissociating the aptamer bound to the binding member in thesolid support/immobilized analyte/binding member complex; (d)translocating the tag through one or more nanopores in a layer; and (e)assessing the tags translocating through the layer, wherein measuringthe number of tags translocating through the layer measures the amountof analyte present in the sample, or wherein detecting tagstranslocating through the layer detects that the analyte is present inthe sample.

Clause 211. The method of clause 210, wherein the immobilization agentcomprises biotin or streptavidin.

Clause 212. The method of clause 211, wherein the immobilization agentcomprises biotin and ligand comprises streptavidin.

Clause 213. The method of clause 211, wherein the immobilization agentcomprises streptavidin and ligand comprises biotin.

Clause 214. The method of any one of clauses 210 to 213, wherein thesolid support, the binding member, and exogenous analyte are addedsequentially or simultaneously to the sample.

Clause 215. The method of any one of clauses 210 to 213, wherein priorto dissociating the aptamer in step (c), the solid support/immobilizedanalyte/binding member complex is isolated.

Clause 216. The method of clause 215 wherein isolation can be done withuse of a magnetic field.

Clause 217. A method for measuring or detecting an analyte present in abiological sample, the method comprising:

I. (a) contacting the sample with a first binding member, wherein thefirst binding member is immobilized on a solid support and wherein thefirst binding member specifically binds to the analyte; (b) contactingthe analyte with a second binding member, wherein the second bindingmember specifically binds to the analyte and wherein the second bindingmember comprises an aptamer; (c) removing aptamer not bound to theanalyte bound to the solid substrate; (d) dissociating the aptamer boundto the analyte and translocating the dissociated aptamer through oracross one or more nanopores in a layer; and (e) assessing the aptamertranslocating through the layer, wherein measuring the number ofaptamers translocating through the layer measures the amount of analytepresent in the sample, or wherein detecting aptamers translocatingthrough the layer detects that the analyte is present in the sample;

II. (a) contacting the sample with a binding member, wherein the bindingmember is immobilized on a solid support and wherein the binding memberspecifically binds to the analyte; (b) contacting the sample with alabeled analyte, wherein the labeled analyte is labeled with a cleavabletag; (c) removing labeled analyte not bound to the binding member; (d)cleaving the tag attached to the labeled analyte bound to the bindingmember; (e) translocating the cleaved tag through or across one or morenanopores in a layer; and (f) assessing the tag translocating throughthe layer, wherein measuring the number of tags translocating throughthe layer measures the amount of analyte present in the sample, orwherein detecting tags translocating through the layer detects that theanalyte is present in the sample;

III. (a) contacting the sample with a binding member, wherein bindingmember is immobilized on a solid support and wherein binding memberspecifically binds to the analyte; (b) contacting the sample with alabeled analyte, wherein the labeled analyte comprises an aptamer; (c)removing labeled analyte not bound to the binding member; (d)dissociating the aptamer bound to the labeled analyte that is bound tothe binding member and translocating the dissociated aptamer through oracross one or more nanopores in a layer; and (e) assessing the aptamertranslocating through the layer, wherein measuring the number ofaptamers translocating through the layer measures the amount of analytepresent in the sample, or detecting aptamers translocating through thelayer detects that the analyte is present in the sample;

IV. (a) contacting the sample with a binding member, wherein the bindingmember specifically binds to the analyte, and the binding member islabeled with a cleavable tag; (b) contacting the sample with aimmobilized analyte, wherein the immobilized analyte is immobilized on asolid support; (c) removing binding member not bound to the immobilizedanalyte; (d) cleaving the tag attached to the binding member bound tothe immobilized analyte; (e) translocating the cleaved tag through oracross one or more nanopores in a layer; and (f) assessing the tagtranslocating through the layer, wherein measuring the number of tagstranslocating through the layer measures the amount of analyte presentin the sample, or detecting tags translocating through the layer detectsthat the analyte is present in the sample; and

V. (a) contacting the sample with a binding member, wherein the bindingmember specifically binds to the analyte, and the binding membercomprises an aptamer; (b) contacting the sample with a immobilizedanalyte, wherein the immobilized analyte is immobilized on a solidsupport; (c) removing binding member not bound to the immobilizedanalyte; (d) dissociating the aptamer bound to the binding member thatis bound to the immobilized analyte and translocating the dissociatedaptamer through or across one or more nanopores in a layer; and (e)assessing the aptamer translocating through the layer, wherein measuringthe number of aptamers translocating through the layer measures theamount of analyte present in the sample, or detecting aptamerstranslocating through the layer detects that the analyte is present inthe sample.

Clause 218. The method of any one of clauses 193 to 217, wherein theratio of the size of the pore to the tag is at or less than 1.0.

1-37. (canceled)
 38. A system for detection of an analyte in a sample,the system comprising: an analyte detection instrument; and one or moreanalyte detection cartridges, wherein the analyte detection instrumentcomprises: a control unit; a detection unit; and a cartridge interfacefor operable connection to the one or more analyte detection cartridges,wherein the control unit is configured for controlling activation of aplurality of electrodes in a cartridge for moving a droplet present inthe cartridge, wherein the detection unit is configured to detect: i) afirst analyte related signal from a droplet in a cartridge; and ii) asecond analyte related signal from a tag or an analyte-specific bindingmember translocating through a pore of a nanopore layer in a cartridge,wherein the each one or more analyte detection cartridge comprises: aplurality of electrodes to generate electrical actuation forces on aliquid droplet in response to the control unit; and a detection regionfor generating an analyte related signal from a droplet in thecartridge; and/or a detection region for generating an analyte relatedsignal from a tag or an analyte-specific binding member translocatingthrough a pore of a nanopore layer in the cartridge.
 39. The system ofclaim 38, further comprising reagent reservoirs, wherein the reagentreservoirs are adjacent to the cartridge interface or are located on thecartridge.
 40. The system of claim 39, wherein the instrument isprogrammed for forming droplets from the reagent reservoirs and formoving the droplets by selectively activating and deactivating theplurality of electrodes.
 41. The system of claim 30, wherein theinstrument is programmed for forming at least a sample droplet from asample introduced into the cartridge and for moving the sampledroplet(s) by selectively activating and deactivating the plurality ofelectrodes.
 42. The system of claim 38, wherein the reagent reservoirscomprise a reagent comprising an enzyme specific for the analyte in thesample, wherein the enzyme acts on the analyte to generate a reactionproduct that generates an electrical or optical signal.
 43. The systemof claim 38, wherein the reagent further comprises a redox mediator. 44.The system of claim 40, wherein the reagent reservoirs comprise a firstbinding member that selectively binds to the analyte, wherein the firstbinding member is attached to a bead.
 45. The system of claim 44,wherein the first binding member is an antibody.
 46. The system of claim44, wherein the reagent reservoirs comprise a second binding member thatselectively binds to the analyte, wherein the second binding member isattached to an enzyme.
 47. The system of claim 46, wherein the secondbinding member is an antibody.
 48. The system of claim 46, wherein theenzyme acts on a substrate to produce a reaction product associated withan electrical signal or an optical signal.
 49. The system of claim 38,wherein the cartridge interface comprises an insertion slot foraccommodating the cartridge, wherein the cartridge comprises: anelectrical detection region, the electrical detection region comprising:a working electrode and a reference electrode for detecting electricalsignal from an electrochemical species generated when an analyte ispresent in the sample; or a nanopore layer disposed between the firstand second substrates, wherein the plurality of electrodes areconfigured for translocating a tag or an analyte-specific binding memberacross the nanopore layer and wherein the electrical detection unitdetects the translocation.
 50. The system of claim 49, wherein theinstrument comprises a plurality of insertion slots.
 51. The system ofclaim 50, wherein the plurality of insertion slots are positioned on acarousel.
 52. The system of claim 38, wherein the system comprisesprogramming for selecting a function of the instrument, wherein thefunction is selected based on type of cartridge operably connected tothe cartridge interface of the instrument, wherein the function isselected from a plurality of assays for processing the sample.
 53. Thesystem of claim 52, wherein when the cartridge comprises an electricaldetection region, the instrument is instructed to process the sample forgenerating an electrical signal in response to presence of an analyte inthe sample and to detect the electrical signal via the electricaldetection unit.
 54. The system of claim 53, wherein instrument receivesan indication of the analyte to be detected and processes the samplebased on the type of analyte to be detected.
 55. The system of claim 54,wherein the indication is provided by a user via a user interface of theinstrument or wherein the indication is provided via a machine-readableindicator present on the cartridge.
 56. The system of claim 38, whereinwhen the cartridge comprises an optical detection region, the instrumentis instructed to process the sample for generating an optical signal inresponse to presence of an analyte in the sample and to detect theoptical signal via the optical detection unit.
 57. The system of claim38, wherein the system is configured for detection of an analyte in awhole blood sample and/or a plasma fraction of a whole blood sample. 58.The system of claim 38, wherein the system is configured for detectionof an analyte in a plasma fraction of a whole blood sample, wherein theplasma fraction of the whole blood sample is generated using a cartridgecomprising a filter for separating the plasma fraction from the wholeblood sample. 59-84. (canceled)